Keywords

1.1 Introduction

Nowadays, plants are the primary sources of food and medicine for mankind. High efficacy and lesser side effects are the main advantage of plant metabolites (Sato and Matsui 2012). Not only that, synthetic molecules are carry various side and adverse effects to our body system (Hesketh et al. 2002). Natural sources contain diversified medicinal component such as alkaloids, glycosides, terpenoids, flavonoids; those are essential for maintaining body immune system. Plants are capable to cultivate a diversified source of natural metabolites; those are important to link up the behavior of other organism. Also, in present time exposure to air pollution, water pollution, ultraviolet ray exposure and deforestation are the most common fact to our nature and our life style. Natural sources are also helps to protect us from these abiotic changes (Hiroaki et al. 2012). In this situation ancient folkloric knowledge of plants and their metabolites is the primary weapon to conquer against various viruses, pathogens, bacteria and their mutant strains. Also in this era different complex structure of secondary metabolites is developed using recombinant DNA technology. As per the definition, metabolites are compounds produced by plants for essential functions (Buchanan et al. 2015); as growth and development (primary metabolites), as well as specific functions as pollinator attraction or defense mechanism. Also metabolites are organic compounds procured from enzyme-dependent chemical reactions of organism known as metabolic pathways (Lena 2012). Nowadays there is a tremendous demand of various plant derived product as health supplement, natural flavors, natural colors, but due to natural calamity and lesser growth of the secondary metabolites the producer does not able to fulfil the demand of the market. Plant tissue culture is one of the way to cope up with the market demand of secondary metabolites (such as alkaloids, glycosides, terpenoids, flavonoids etc.) and helps to over produce the secondary metabolites (Pichersky and Gang 2000). Actually plant tissue culture comprised of clonal propagation, callus formation and formation of germplasm using a perfect combination of plant hormones, temperature, humidity and elicitors. Tissue culture is not only helps to generate secondary metabolites but also participate in seed germination, improvement of crop quality, immobilization, biomass accumulation, micropropagation and existence of some rare plants (those are lost during evolution) (Jensen et al. 2014). Tissue culture mainly focused on shortening of biosynthetic process of plant cell, higher cell division and metabolism; these all factors are cumulatively increase the growth of secondary metabolites. This chapter mainly focus on the role of elicitors on the production of secondary metabolites.

1.2 Types of Plant Metabolites

Plant metabolites were two types such as primary metabolite and secondary metabolite.

  1. 1.

    Primary metabolites were the basic requirement for growth and development and it was present in all plants, organisms or cells. Low molecular weight primary metabolites were important components of crop plants for both consumers and producers. Primary metabolites such as ethanol, lactic acid, and certain amino acids were the most primary one. Primary metabolism was referred as trophophase, which was characterized by balanced growth of microorganisms. It occurred when all the nutrients needed by the organisms were provided in the medium. Primary metabolism was essential for plant existence and reproduction of cells. In the trophophase, the cells possessed with optimal concentrations of all the macromolecules such as proteins, DNA, RNA etc. In during trophophase, growth of microorganisms was followed the exponential nature. During trophophase, the collective metabolic products were known as primary metabolites (Croteau et al. 2000).

    Basically primary metabolites were subdivided into two groups, such as:

    1. a.

      Primary essential metabolites: These were the compounds produced in adequate quantizes to sustain cell growth e.g. vitamins, amino acids, nucleosides. Primary microorganisms usually do not overproduce essential primary metabolites because it was wasted. In case of industrial overproduction, the regulatory mechanisms were taken.

    2. b.

      Primary metabolic end products: These were the normal and traditional end products of fermentation process of primary metabolism. The end products had many industrial applications such as in the form of ethanol, acetone and lactic acid. Carbon dioxide was a metabolic end product of Saccharomyces cerevisiae, which was essential for leavening of dough in baking industry.

    Growth limitation: Due to insufficient supply of any nutrient (substrate or even Oxygen), the growth of microorganisms was slows down. However, the metabolism does not stop, continues until cell lives with the differentiate product formation (Pal et al. 2019a).

  2. 2.

    Secondary Metabolites: These are sometimes colored, fragrant and flavorful compounds, which typically conjugate with plant metabolite and organisms. Interactions were included pollination through animal such as butterfly, bees, moths, flies as well as by fungi, bacteria, nematodes, and viruses and by foliage, gastropods and caterpillar. After the ending of exponential growth of microorganisms, idiophase was initiated. Idiophase was characterized by secondary metabolism. Secondary metabolites (produced in abundance) were not required by the microorganisms, but it had some industrially very important mainly in the biotechnology to produce antibiotics, steroids, alkaloids, glycosides, plant hormones and toxins (Pal et al. 2017).

    Characteristics of secondary metabolites:

    1. a.

      Secondary metabolites were specifically produced by selected microorganisms.

    2. b.

      Secondary metabolites were less essential for development and reproduction of organisms.

    3. c.

      Environmental factors were influenced the production of secondary metabolites (Pal et al. 2019b).

    4. d.

      Certain microorganisms can produce structurally related secondary metabolites as a group of compounds not a single one such as anthracyclines, produced by Streptomyces.

    5. e.

      The biosynthetic pathways of were not clearly established for most secondary metabolites.

    6. f.

      The regulation of the formation of secondary metabolites was more complex than primary metabolites (Pal et al. 2018).

    Functions of secondary metabolites: There were two possible hypotheses for the justification of secondary metabolite function as:

    1. a.

      Secondary metabolites were beneficial for the cells to survive.

    2. b.

      The secondary metabolites were important for the cell development.

    The metabolic regulation was equally complex to achieve overproduction of secondary metabolites. Some regulatory mechanisms were as follows:

    1. i.

      Induction: Methionine addition induced certain enzymes, which enhanced the production of cephalosporin. Also tryptophan regulated ergot alkaloid biosynthesis.

    2. ii.

      ii. End product regulation: Secondary metabolites inhibited their own biosynthesis by negative feed regulation such as penicillin, streptomycin, puromycin and chloramphenicol (Pal et al. 2019c).

    3. iii.

      Catabolite regulation: In this process, a key enzyme was participated in the catabolic pathway was inactivated, inhibited or repressed the process. Catabolic repression was obtained by carbon or nitrogen sources. Where the most common source of carbon was glucose, which was participated in the inhibition process of several antibiotics, such as: penicillin, streptomycin, bacitracin, chloramphenicol, puromycin; whereas ammonia was the primary source of nitrogen which was catabolite regulators for the overproduction of certain antibiotics (Pal et al. 2019d).

    4. iv.

      Phosphate regulation: Inorganic phosphate was essential for the growth and multiplication of prokaryotes and eukaryotes. Concentration of inorganic phosphate (up to 1 mM) was directly correlated with the concentration of secondary metabolites e.g. streptomycin, tetracycline, alkaloids, gibberellins.

    5. v.

      Auto regulation: Certain microorganisms such as: actinomycetes were associated with the self-regulation for the production of secondary metabolites. A compound (factor A) was a derivative of a hormone was suggested to be closely involved in auto regulation for the production streptomycin by Streptomyces griseus.

    6. vi.

      Bioconversions: Biotransformation process using microorganisms was very important for the production of several compounds e.g. vinegar, sorbose, steroid hormones and certain amino acids. In this process, microorganisms were responsible for the conversion of a compound to another structurally related product in one or a few enzymatic reactions. The bioconversions can be performed with resting cells, spores or even killed cells. Non-growing cells were preferred for bioconversions due to its high substrate concentration (Pal et al. 2019e).

1.3 Elicitor and Its Type

Elicitors are the substances which under stress conditions induce the biosynthesis of secondary metabolites of plants. So elicitation is the process to create stress on the plant, which relates with the chemical composition and growth of the plant. The process of elicitation mainly increase the growth of the plant and its metabolites.

Elicitors are two types such as abiotic and biotic elicitor. Abiotic elicitors are obtained from non-living source. Abiotic elicitors are mainly three types such as: physical, chemical and hormonal abiotic elicitors. Biotic elicitors are obtained from biological source and it has four types such as: saccharides, yeast, fungal and bacterial biotic elicitors (Fig. 1.1).

Fig. 1.1
figure 1

Elicitor and its types

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1.4 Application of Elicitors on the Production of Secondary Metabolites

1.4.1 Production of Secondary Metabolites Using Abiotic Elicitors

1.4.1.1 Effect of Arachidonic and Jasmonic Acid Elicitors on Secondary Metabolites of Wheatgrass

Zlotek et al. was scientifically tested the effect of abiotic elicitors as arachidonic acid and jasmonic acid on the phenolic and flavonoid content of wheatgrass (Triticum aestivum L.). The experiment started with slightly grown wheatgrass plant treated with different concentration of arachidonic acid and jasmonic acid (arachidonic acid: 0.01, 1.0 and 100 µM; jasmonic acid: 0.01, 1 and 100 µM) at a relative humidity of 70%, 18 °C of temperature and 200 µmol m−2 s−1 of photon flux density. Thereafter the growth of the plants were prominent, then make it dry followed by centrifuged at 9000 g force for 30 min. The total phenolic content, flavonoid content, Antioxidant and Anti-inflammatory activities were evaluated. The outcomes revealed that amount of principle flavonoids (luteolin and apigenin) and phenolic compounds (ferulic acid and syringic acid) were remain unchanged after elicitation, some polyphenolic compounds observed with increased amount; but the anti-inflammatory activity was markedly improved. The outcomes concluded that 0.01 μM of arachidonic acid showed better elicitation behavior than jasmonic acid (Złotek et al. 2019).

1.4.1.2 Effect Abiotic Elicitors on Secondary Metabolites of Thyme, Greater Celandine and Parsley

Kleinwachter et al. was experimentally proved the effects of slight drought and abiotic elicitors on the production of secondary metabolites of thyme (Thymus vulgaris), greater celandine (Chelidonium majus) and parsley (Petroselinum crispum). The experimental procedure was started with the minimization of water supply to the plant by 50% and continue until the evaporation done by plants were 80% reduced than usual. These conditions leads to the abrupt ratio of soil and water by (6–10)% as compare to the control plant (17–20)%. Then the samplings of thyme and greater celandine were treated with methyl jasmonate (MJ) (0.2 mM) and parsley sampling was treated with (2 mM) concentration, followed by addition of salicylic acid solution. Then the samplings were quantified the production of monoterpenes in thyme, benzylisoquinoline alkaloids in greater celandine, flavones and essential oil in in parsley. The outcomes revealed that the quantity of benzylisoquinoline in greater celandine was increased by 46% and flavones were increased by 70% in parsley; but the minimized water supply reduced the overall content of the plants due to its lesser growth. The presence of salicylic acid does not create any remarkable effect and also the different ratios of MJ regulated the plant secondary metabolites growth depends upon their species characteristics (Kleinwachter et al. 2015).

1.4.1.3 Effect of Abiotic Elicitor on Triterpenoid Accumulation in Centella asiatica

Buraphaka et al. was scientifically tested the effects of abiotic elicitor (MJ and salicylic acid) on the accumulation of triterpenoid in Centella asiatica plant. The experiment was started with the reaction between different concentrations of MJ and salicylic acid (1, 2 and 4 mM) with the leaves of the plant under a shaker for 20 min, 40, 60 and 120 min. Then the treated leaves were divided into two groups: first group for anti-inflammatory was dried at 50 °C whereas another group was stored at (−) 80 °C for messenger RNA level expression analysis. The outcomes revealed that after the treatment with salicylic acid (2 mM), the amount of triterpenoid was doubled; whereas treatment with MJ the amount of increased content was 1.4 times than normal. Also the elicited leaves were remarkably inhibited the nitric oxide production in lipopolysaccharide induced RAW 264.7 macrophage cells with increased activity of phenylalanine ammonia lyase, peroxidase and catalase enzymes. So these data correlated with the positive effect of abiotic elicitor on the aforementioned plant (Buraphaka and Putalun 2020).

1.4.1.4 Effect of Abiotic Elicitor on Affinin Contents in Heliopsis longipes (Chilcuague)

Parola-Contreras et al. was experimentally proved the effects of salicylic acid and hydrogen peroxide as aboiotic elicitor on the amount of affinin content in Heliopsis longipes (chilcuague). The experiment was started with the treatment of the samplings of chilcuague and salicylic acid (5 and 10 mM) at 150 days after transplanting as well as hydrogen peroxide (200 and 400 mM) at 157 days after transplanting. Then the elicited plants were evaluated with the enzymatic activity for superoxide dismutase, catalase, phenylalanine ammonia lyase, valine decarboxylase and also quantified the amount of affinin present in the plant. The outcomes revealed that after 150 days maximum superoxide dismutase, catalase, phenylalanine ammonia lyase and valine decarboxylase activities were observed with 10 mM of salicylic acid, 200 mM of hydrogen peroxide, both salicylic acid and hydrogen peroxide, 200 mM of hydrogen peroxide as abiotic elicitor. After 157 days, maximum superoxide dismutase, catalase, phenylalanine ammonia lyase were observed with 10 mM of salicylic acid whereas maximum valine decarboxylase activity was observed with 200 mM of hydrogen peroxide elicitor (Fig. 1.2). After 150 and 157 days, maximum affinin content was observed with 10 mM of salicylic acid and 200 mM of hydrogen peroxide. These data confirmed the positive effects of abiotic elicitors on chilcaugaue (Parola-Contreras et al. 2020).

Fig. 1.2
figure 2

Copyright permission obtained from Parola-Contreras et al. @ 2020 Elsevier B.V

Typical morphological aspect at day 164 post-transplanting of H. longipes treated with hydrogen peroxide and salicylic acid.

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1.4.1.5 Effects of Abiotic Elicitor on Hypocrellin a Content in Shiraia bambusicola

Lu et al. was scientifically proved the effect of abiotic elicitor (lanthum La3+) on the content of hypocrellin A (anticancer agent mainly against lung adenocarcinoma) present in Shiraia bambusicola (parasitic fungi present in bamboo). The experiment was started with mycelium of S. bambusicola and lanthum (La3+) with a concentration range from (0.0–1.4 g/L). The elicited fungus was evaluated by membrane permeabilization assay with fluorescent dye SYTOX green, generation of reactive oxygen species using 7-dichloro dihydro fluoresceindiacetate dye followed by antioxidative activity using NADPH oxidase produce superoxide, superoxide dismutase and catalase enzymes. The outcomes showed that after lanthanum treatment for 4 h, the hyphae became more strong and intense. The bioactivities showed that after lanthum treatment the amount of hydrogen peroxide was higher with many fold increased in antioxidative enzymatic activity. Also vitamin C suppressed the up regulation of major facilitator superfamily transporter and O-methyltransferase, polyketide synthase, ATP-binding cassette transporter, O-methyltransferase/FAD-dependent monooxygenase and FAD/FMN-dependent oxidoreductase genes. These data correlated with the greater production of hypocrellin A in lanthanum elicitor induced fungi (Lu et al. 2019a).

1.4.1.6 Effects of Abiotic Elicitor on Steviosides Production in Stevia rebaudiana Bertoni Calli

Mejia-Espejel et al. was experimentally proved the effects of abiotic elicitor (salicylic acid, MJ, citric acid, ascorbic acid) with different light (red, blue and white) and temperature environment on the production of steviosides (diterpene glycoside) present in Stevia rebaudiana. The experiment was started with the reaction between 3 gm of calii (grown in Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid, BA, citric acid and ascorbic acid) and different elicitors such as salicylic acid (10 and 100 mM), MJ (10 and 100 mM), antioxidants, growth regulator, different light (white light, red light, blue light and a combination of red light and blue light) and temperature (25 °C and 28 °C) as physical abiotic elicitor. The outcomes revealed that maximum stevioside was obtained at a combination of white light, 28 °C, 100 mM of salicylic acid and 10 mM of MJ. These data strictly adhere with the importance of abiotic elicitor on the production of steviosides (Mejia-Espejel et al. 2018).

1.4.1.7 Effect of Light as Abiotic Elicitor on the Secondary Metabolite Growth in Stevia rebaudiana

Ahmad et al. was experimentally proved the effect of light of the production of secondary metabolites (total phenolic and flavonoid content) and antioxidative properties of the Stevia rebaudiana plant. The experiment was started with the development of callus from the leaves of the plant in MS medium contained with 2,4-dichlorophenoxyacetic acid, BA; then the callus was exposed to different light medium such as green light, yellow light, blue light, red light and white fluorescent light environments at a 25 °C temperature flowed by antioxidative property assessment using DPPH antioxidant method. The outcomes revealed that maximum fresh callus weight was observed in white light with greater growth kinetic. Total phenolic and flavonoid contents were observed in blue light (Fig. 1.3). Also blue light elicitor increased the antioxidative property of the callus. These data confirmed the importance of blue light on the growth of secondary metabolites of Stevia rebaudiana (Ahmad et al. 2016).

Fig. 1.3
figure 3

Copyright permission obtained from Ahmad et al. @ 2016 Elsevier B.V

Effect of different spectral lights on callus morphological features in S. rebaudiana. a red light induced callus b blue light c yellow light d green light and e control white light.

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1.4.1.8 Effect of Gamma Radiation on Secondary Metabolites of Hypericum triquetrifolium Turra

Azeez et al. was scientifically proved the effect of abiotic elicitor (gamma radiation) on the production of biomass and secondary metabolites (phenolic compounds and naphtodiantrones) in Hypericum triquetrifolium Turra plant. The experiment was started with induction of explants (obtained from leaf, stem and root) with indole acetic acid (IAA) and thidiazuron (synthetic cytokinin) in MS medium followed by irradiated with gamma rays of 10, 20, 30 and 40 Gy unit; then finally quantify its average growth index based on the values of callus biomass at initial and final point. The outcomes revealed that at 10 Gy scale maximum growth index was observed as well as maximum accumulation of 4-hydroxybenzoic acid, chlorogenic acid and epicatechin. At 10 Gy of gamma irradiation the accumulation of naphtodiantrones (hypericina and pseudohypericin) were observed (Fig. 1.4). So it was quite resemble that 10 Gy scale of gamma rays directly helps to accumulate the principle secondary metabolites present in the plant (Azeez et al 2017).

Fig. 1.4
figure 4

Copyright permission obtained from Azeez et al. @ 2017 Elsevier B.V

The morphological characteristics of Hypericum triquetrifolium Turra callus mass accumulated from leaf explant and irradiated with 10 Gy dose after the third successive subculture (a). Hypericum triquetrifolium T. callus mass from leaf irradiated with 30 Gy dose and harvested after the third regular subculture (b).

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1.4.1.9 Effects of Abiotic Elicitors on Secondary Metabolites of Vitis vinifera Suspension Culture

Cai et al. was experimentally proved the importance of abiotic elicitor (streptomycin, activated charcoal, etephon) and pressure on the growth of secondary metabolites of Vitis vinifera plant. The experiment was started with the reaction between plant cell cultures with the above mentioned abiotic elicitors along with a diverse pressure treatment from (40–50) Mega Pascal unit for a seven days regime. Here streptomycin was used to reduce the load of contamination, activated charcoal was utilized as source of carbon and etephon was a plant growth regulator. The outcomes revealed that after 5th day of treatment maximum fresh and dry weight of the callus was observed with activated charcoal and etephon treatments. Also the amount of anthocyanins were increased with etephon and activated charcoal treatments. But the amount of extracellular 3-O-glucosyl resveratrol and phenolic compound were increased with the combination treatment of etephon and high pressure. These data confirmed the importance of abiotic elicitors on the growth and accumulation of secondary metabolites present in Vitis vinifera plant (Cai et al. 2011).

1.4.1.10 Effect of Salicylic Acid on Alkaloid Biosynthesis in Marine Microalgae Arthrospira platensis

Hadizadeh et al. experimentally proved the importance of salicylic acid as abiotic elicitor on the biosynthesis of pharmaceutical alkaloid present in marine microalgae Arthrospira platensis. The experiment as started with addition of salicylic acid (0, 5, 20 and 100) µM concentration to A. platensis suspension culture after 3, 7, 10 and 14 days of culture; followed by estimation of dry weight of biomass and total alkaloid content. The outcomes revealed that maximum growth was observed between 8 to 10 days of culture and at 15 days maximum dry weight of biomass was observed with salicylic acid (5 and 20) µM as well as at 15th day of treatment maximum content of alkaloid was obtained with 5 µM of salicylic acid. These data confirmed the importance of salicylic acid on the production of alkaloid in the marine microalgae (Hadizadeh et al. 2019).

1.4.1.11 Effect of Abiotic Elicitor on Growth of Secondary Metabolites in Broccoli Plant

Hassini et al. scientifically proved the importance of abiotic elicitors (salicylic acid, MJ and methionine) on the growth of secondary metabolites present in broccoli plant. The experiment was started with treating the seeds with potassium chloride, potassium sulfate and sodium chloride followed by elicited the roots and shoots with methionine (10 mM), salicylic acid (200 µM) and MJ (100 µM) concentrations. The capacity of roots to conduct water between roots to xylem was measured by root hydraulic conductivity using root fresh weight and pressure, followed by assessment of plant defense mechanism using myrosinase activity and total phenolic content present in the plant was also evaluated. The outcomes showed that maximum dry weight of plant was obtained from roots using potassium sulfate and from shoots using methionine as elicitor. The root hydraulic conductivity showed that methionine positively culminated the plant. The myrosinase activity showed that methionine increase the defense mechanism while sodium chloride, salicylic acid were suppressed the defense of the plant against insect and herbivores. Two phenolic acid component as chlorogenic acid and sinapic acid were increased in presence of potassium chloride and potassium sulfate whereas flavonol was maximum obtained through methionine elicitation. These data confirmed the importance of abiotic elicitors on the growth of secondary metabolites present in broccoli plant (Hassini et al. 2019).

1.4.1.12 Effect of Abiotic Elicitors on Steviol and Adventitious Root Growth in Stevia rebaudiana Plant

Kazmi et al. scientifically proved the importance of abiotic elicitors (MJ, phenyl acetic acid and melatonin) on the growth of steviol glycoside and adventitious roots in Stevia rebaudiana plant. The experiment was started with culturing the small pieces of root, stem and leaf portions of the plant in MS medium with BA, IAA and naphthalene acetic acid (NAA) as growth regulators, then the explants were elicited using different concentrations of melatonin, phenyl acetic acid and MJ for a period of 15, 30 and 45 min. The elicited dry mass of the explants were estimated with total phenolic and flavonoid content as well as evaluated by antioxidative assay methods. The outcomes revealed that in case of root maximum morphogenic response was given by combination of 2.0 mg/L of benzylaminopurine (BA) and 1.0 mg/L of NAA; in case of leaf maximum morphogenic response was obtained from 0.5 mg/L of NAA and from stem maximum morphogenic response was obtained using 2.0 mg/L of BA plant regulators. Maximum adventitious roots were obtained from 0.5 mg/L of MJ after 30 min dipping time. Total phenolic and flavonoid contents were highly obtained from in vitro plants but among the elicitors MJ-adventitious root combination wins the race and MJ-adventitous root combination was also observed with higher antioxidative effect (Fig. 1.5). So these data confirmed the importance of the abiotic elicitors on Stevia rebaudiana plant (Kazmi et al. 2019).

Fig. 1.5
figure 5

Copyright permission obtained from Kazmi et al. @ 2019 Elsevier B.V

In vitro morphogenesis and adventitious root (AR) formation a In vitro germinated plants, b and c response of root and stem explants to 6-benzyladenine (BA), d–f different morphological responses by leaf explants, d callus formation, e AR induction from callus, f direct AR formation, g–j AR formation in leaf explants pretreated with elicitors for defined time periods, g Melatonin (Mel) induced AR, h: Phenyl acetic acid (PAA) induced AR, i and j Methyl jasmonate (Me-J) induced AR.

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1.4.1.13 Effect of Ultrasound on Secondary Metabolite Accumulation in Tomato Plant

Lu et al. experimentally proved the importance of high intensity ultrasound on secondary metabolite accumulation and antioxidative efficiency of Solanum lycopersicum (tomato) plant. The experiment was started with ultrasound treatment of tomatoes with ultrasound (25 kHz) for (1–4) min at room temperature with 26 W/L of acoustic power density. After 0 h, 24 h and 48 h of treatment, the elicited tomatoes were checked for firmness, total phenolic content, lycopene content, total carotenoid content and ascorbic acid followed by antioxidative efficiency evaluation. The outcomes showed that firmness of tomatoes were not remarkably changed so it can stored for longer time; total phenolic content was maximum after 48 h of storage with 2 min of ultrasound treatment; maximum lycopene and carotenoid contents were obtained after 48 h of storage with (1–4) min of treatment as well as ascorbic acid content was higher with 3 min of ultrasound treatment after 48 h. Maximum phenylalanine ammonia-lyase activity was also observed after 48 h of storage with (1–4) min of ultrasound treatment. These data confirmed the importance of abiotic elicitors on secondary metabolite accumulation in tomato plant (Lu et al. 2020).

1.4.1.14 Effect of Abiotic Elicitors on Metabolite Accumulation of Trifolium resupinatum

Twaij et al. experimentally proved the importance of abiotic elicitors and precursors on the accumulation of secondary metabolites in Trifolium resupinatum. The experiment was started with culture propagation of T. resupinatum shoot in MS medium containing BA (2.0 mg/L), IAA (0.5 mg/L), sucrose and phytage at pH 7.0 until calli was full grown. Then the calli further transferred into the MS medium with sucrose, NAA and kinetin as growth regulators. Then the grown explants were divided into four categories such as shade grown plant, somatic embryos, light induced and dark induced followed by treated with MJ, salicylic acid and glutathione with concentration range (0, 10, 20, 40, 80 and 160) µM for a period of (0–4) weeks. Then total phenolic content, total flavonoid and antioxidative efficiency using DPPH radical scavenging and ferric reducing antioxidant potential assay activity were checked and evaluated for/by elicited explant. The outcomes revealed that for shade grown plant, somatic embryos and light induced and dark induced conditions antioxidative efficiencies against DPPH and ferric reducing antioxidative processes were observed with greater efficiency in MJ, salicylic acid and glutathione elicitors. Maximum effects as antioxidative and enzymatic activities against glutathione peroxidase, glutathione reductase and glutathione-S-transferase were observed with salicylic acid as elicitor. These data correlated abiotic elicitor and secondary metabolites accumulation in Trifolium resupinatum plant (Twaij et al. 2019).

1.4.1.15 Effect of MJ Abiotic Elicitor on Anthraquinone Production in Rubia tinctorum

Perassolo et al. experimentally proved the effects of combined culture medium and MJ as abiotic elicitor on the production of anthraquinone in Rubia tinctorum plant. The process was started with culturing of young small clones of leaf margin or aerial stem of the plant with Agrobaterium rhizogenes in Gamborg B5 medium with sucrose and ampicillin as source of carbon and strengthening of natural defense system. Then after two to four weeks roots were developed for cloning in Gamborg B5 and Llyod-McCown woody plant medium with thiamine, pyridoxine, nicotinic acid and myoinositol as growth regulators. After fourteen days, inoculum cultures were elicited in presence of MJ (100 µM) concentration. After a time interval of 0, 2, 4 and 7 days, the amount of anthraquinone and growth of biomass were evaluated. The outcomes revealed that after six weeks of treatment biomass in woody plant medium was higher than Gamborg medium and for anthraquinone production Gamborg medium was perfectly suited. But after seven days treatment with MJ, anthraquinone content was remarkably good as compare to dimethylsulfoxide. These information stated the importance of MJ and growth regulators for the production of anthraquinone (Perassolo et al. 2017).

1.4.1.16 Effect of Polyunsaturated Fatty Acids on Gymnemic Acid Production in Gymnema Sylvestre

Praveen et al. scientifically proved the importance of polyunsaturated fatty acids (oleic acid and linoleic acid) on the production of gymnemic acid present in Gymnema sylvestre plant. The experiment was started with formation of hairy root culture of the plant with the plantation in MS medium with sucrose as carbon source and these were sub-cultured for fortnight period. After the incubation period, cultures were elicited with oleic acid and linoleic acid with concentration range (0, 1, 5, 10 and 50) µM. The dry biomass, amount of gymnemic acid, total phenolic content, total flavonoid content were checked as well as antioxidative property was also evaluated. The outcomes revealed that the growth ratio between fresh and dry biomass was higher with linoleic acid (1.0 µM) elicitation. Highest amount of gymnemic acid and greater antioxidative property were observed with linoleic acid (5.0 µM) elicitation. These data confirmed the importance of linoleic acid for the production of gymnemic acid (Praveen et al. 2014).

1.4.1.17 Effect of Magnesium Oxide Nanoparticle on Secondary Metabolite in Atropa belladonna

Tian et al. experimentally proved the importance of magnesium oxide nanoparticle on the growth and accumulation of antioxidative metabolites present in Atropa belladonna plant. The experiment was started with germination of the plant in MS medium containing sodium hypochlorite, then the clones of root and shoot were elicited with magnesium oxide nanoparticle of (25, 50, 100 and 200) mg/L concentration without any presence of cytokinin and auxin. Then the shoot/root number, length and fresh weights were evaluated; evaluation of relative water content, chlorophyll content, malondialdehyde and membrane stability index, enzymatic activities against superoxide dismutase, ascorbate peroxidase were also evaluated. Also the total phenolic flavonoid content, total alkaloid and antioxidative efficiencies were measured. The outcomes revealed that shoot/root number, length and fresh weight, relative water, chlorophyll and membrane stability index were high with 25 mg/L of magnesium oxide nanoparticle presence. Total phenolic and flavonoid contents were observed with (100 mg/L) magnesium oxide nanoparticle; maximum alkaloid content was observed with (25 mg/L) of nanoparticle was well as maximum antioxidative effect of the elicited plant was observed with (200 mg/L) magnesium oxide nanoparticle (Tian et al. 2018).

1.4.1.18 Effect of Temperature on Secondary Metabolite Accumulation in Cold Environment Soil Fungi

Ulaganathan et al. experimentally suggested the effect of temperature on secondary metabolite accumulation in forty soil isolated fungal strains. The experiment was started with cultivation of isolated fungi into potato dextrose agar plate for the mycelia formation followed by antimicrobial assessment against gram positive (Bacillus subtilis, Enterococcus facaellis and Bacillus cereus) and gram negative (Pseudomonas aeruginosa and Escherichia coli) strains. As per the first screening test, the pass over fungal strains [HND 10 (Atradidymella sp), AK 102 (Pseudogymnoascus sp.) and HND 11 (Penicillium flavigenum)] were evaluated against bacterial strains (E. coli, B. subtilis, S. aureus, P. aeruginosa and Candida albicans) at different temperature modules as 4, 10, 15 and 28 °C. The outcomes showed that the growth of E. coli, B. subtilis, S. aureus and C. albicans were highly inhibited by AK 102 at 4 and 15˚C temperature; P. aeruginosa was not observed with any susceptibility against strains. So it was quite justified the importance of temperature on microbial growth inhibition (Ulaganathan et al. 2017).

1.4.1.19 Effect of Polyunsaturated Fatty Acids on Secondary Metabolite Production in Panax ginseng

Wu et al. experimentally proved the effect of polyunsaturated fatty acids (linoleic and alpha linolenic acid) on secondary metabolite accumulation and biomass production of Panax ginseng plant. The experiment was started with culturing of adventitious root of ginseng into MS medium containing indole butyric acid and sucrose. When the roots were 5 fresh weight per litre, these were inoculated into the same medium with addition of linoleic acid and alpha-linolenic acid with concentrations of (1.0, 2.5, 5.0, 10.0 and 20.0) μmol per litre. Total ginsenosides, diol ginsenoside, triol ginsenoside, total phenolic and total flavonoid contents were checked for polyunsaturated acid elicited ginseng plant adventitious roots. The outcomes showed that maximum amount of total ginsenosides, diol ginsenoside, triol ginsenoside and total flavonoid contents were observed with 5.0 μmol/l of fatty acid elicitation, whereas total phenolic content was maximum with 5.0 μmol/l of linoleic acid and 10 μmol/l of alpha linolenic acid. The enzymatic activity data expressed that maximum superoxide dismutase activity was observed with 20 μmol/l of linoleic acid and 5.0 μmol/l of alpha linolenic acid, maximum catalase activity was obtained with 2.5 μmol/l of linoleic acid and 5.0 μmol/l of alpha linolenic acid, maximum ascorbate peroxidase activity was obtained with 20 μmol/l of linoleic acid and 5.0 μmol/l of alpha linolenic acid as well as glutathione peroxidase was higher in case of 2.5 μmol/l of linoleic acid and alpha linolenic acid. These data confirmed the importance of fatty acids on the secondary metabolite production and antioxidative properties of ginseng plant (Wu et al. 2009).

1.4.2 Production of Secondary Metabolites Using Biotic Elicitors

1.4.2.1 Plant Growth Regulating Rhizobacteria Stimulated Secondary Metabolite Growth in Pennyroyal

Asghari et al. experimentally proved the effect of plant growth regulating rhizobacteria on the biosynthesis of secondary metabolites in pennyroyal (Mentha pulegium) plant under droght situation. The experiment was started with culturing the seeds of pennyroyal plant with rhizobacteria in four different categories such as control group without rhizobacteria, treated with Azotobacter chroococcum, treated with Azospirillum brasilense and final group treated with a combination of A. chroococcum and A. brasilense followed by treat in the environment with enough water, moderate water and less water condition. Relative water content, chlorophyll fluorescence, total phenolic content, total flavonoid content, antioxidative activities using glutathione peroxidase, catalase and superoxide dismutase and radical scavenging activity against DPPH were checked and evaluated by the elicited plant. The outcomes showed that relative water and chlorophyll contents were highest in enough water condition and lowest with less water condition; the glutathione peroxidase, catalase and superoxide dismutase activities were maximum less water condition with A. chroococcum elicitated condition followed by A. brasilense and its combination. Abscisic acid content, total phenolic content, total flavonoid content, radical scavenging activities as well as presence of essential oils such as 1,8-cineloe, menthone and pulegone were maximum in case of less water condition with A. chroococcum and A. brasilense combination; whereas piperitone was maximum obtained with moderate water condition with A. chroococcum and A. brasilense combination. So these data directly stated the importance of rhizobacteria on pennyroyal plant (Asghari et al. 2020).

1.4.2.2 Effect of Carrageenan on Secondary Metabolite Growth in Chickpea and Maize Plant

Bi et al. scientifically proved the importance of carrageenan (polysaccharide) on the growth of secondary metabolite present in chick pea and maize plants. The process was started with formation of two types of elicitors as liquid carrageenan elicitor of 100 µg glucose concentration and solid carrageenan elicitor (mixer of hot aqueous extract of carrageenan with soil). The treatment module had three types: (i) 50 ml of liquid elicitor (ii) 5 g of solid elicitor (iii) 5 ml of liquid elicitor. These modules were applied to both chickpea and maize plants. Plant height, number of pods/branches/leaves per plant, and plant height, number of cobs per plant, stem diameter, number of leaves per plant, days to flowering, secondary metabolite production were evaluated for maize plant. Maximum results were obtained from treatment with 50 ml of liquid elicitor. So these data confirmed the importance of carrageenan in liquid form for the growth and production of secondary metabolite present in chickpea and maize plants (Bi et al. 2011).

1.4.2.3 Effect of Fungal Elicitor on Phenylalanine Ammonia Lyase Activity in French Bean Cells

Bolwell et al. scientifically proved the importance of fungal pathogen obtained from Colletotrichum lindemuthianum fungal pathogen on phenylalanine ammonia lyase activity of cultured french bean cells. The experiment was started with treatment of cultured french bean cells with calcium ionophore, calmodulin inhibitor (trifluroperazine), calcium channel blocker (verapamil), pertussis toxin, cholera toxin, polyether antibiotic (monensin), labdane diterpene (forskolin), local anesthetic (procaine), caffeine and fungal elicitor. The outcomes revealed that on the activity of elicitor, caffeine and procaine had no direct effect whereas most effect on elicitation was observed with ionophore and most negative effect on elicitation was observed with monensin (Bolwell et al. 1991).

1.4.2.4 Effect of Endophytic Fungi on Secondary Metabolite Accumulation in Rumex gmelini Turcz

Ding et al. scientifically proved the importance endophytic fungi (Aspergillus sp., Fusarium sp., and Ramularia sp.) on the secondary metabolite accumulation of R. gmelini Turcz plant. The experiment was started with the development of explant using rhizomes of the plant in MS medium. After fifteen days, roots were co-cultured with endophytic fungi with three concentration range (1000, 10,000 and 100,000 mL−1) followed by measured the amount of secondary metabolites (polydatin, resveratrol, chrysophaein, musizin, emodin, chrysophanol and physcion) accumulation. The outcomes revealed that Aspergillus sp. was used to increase the production of resveratrol, chrysophaein, musizin, emodin, chrysophanol and physcion whereas Fusarium sp. was used for polydatin. In case of combination treatment, R. gmelini seedling cultured with three endophytic fungi (Aspergillus sp. = 1000 mL−1; Fusarium sp. = 10,000 mL−1 and Ramularia sp. = 100,000 mL−1) for a period of 20 days created positive effects on polydatin, resveratrol, chrysophaein, musizin, chrysophanol and physcion whereas productivity of emodin was increased many folds with combination of R. gmelini seedling cultured with two endophytic fungi Aspergillus sp. = 1000 mL−1; Fusarium sp. = 10,000 mL−1 after 15 days of treatment. These data cumulatively stated the importance of endophytic fungi on the production of secondary metabolites present in R. gmelini (Ding et al. 2018).

1.4.2.5 Effect of Chitosan on Flavonoid Productivity in Isatis tinctoria L. Hairy Root Cultures

Jiao et al. experimentally proved the importance of chitosan on the increased production of flavonoid and antioxidative efficiency of Isatis tinctoria L. The experiment was started with formation of hairy root of I. tinctoria in MS medium with sucrose as principle constituent. After 24 days, chitosan in acetic acid (concentration = 50, 100, 150, 200 and 400 mg/l) were added to hairy roots of the plant followed by incubated for a period of 0, 6, 12, 18, 24, 30, 36, 48, 60, 72, and 96 h. Total flavonoid content especially (rutin, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol, and isoliquiritigenin) were checked after the chitosan treatment. The outcomes revealed that total flavonoid content was increased with increasing concentration of chitosan (upto 200 mg/l); incase of 400 mg/l chitosan, the productivity of total flavonoid was decreased. Among other flavonoids, rutin, followed quercetin and isorhamnetin were highly expressed (Fig. 1.6). Antioxidative efficiencies of elicited hairy root cultures of I. tinctoria (150 mg/l) were good as compare to ascorbic acid (as per percent radical scavenging activity) and butylated hydroxytoluene (as per percent bleaching inhibition activity). These data confirmed the effects of chitosan on I. tinctoria secondary metabolite production (Jiao et al. 2018).

Fig. 1.6
figure 6

Copyright permission obtained from Jiao et al. @ 2018 Elsevier B.V

Phenotype comparison of hairy root tissues from control and ITHRCs elicited by 150 mg/L chitosan for 36 h.

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1.4.2.6 Effect of Biotic Elicitor on Glyceollin in Soybean (Glycine max)

Kalli et al. scientifically proved the cumulative effects of reactive oxygen species and a mixture of fungal strains (Rhizopus oligosporus and Rhizopus oryzae) on the production of natural antimicrobial prenylated pterocarpan glyceollin and isoflavonoid present in soybean plant. The experiment was started with the formation of seedling using two different cultivar (I and II) in three phase (soaking, germination and priming). Soaking was done for one day followed by germination for two days. Priming was done by two phase such as early priming with reactive oxygen species and stress priming with slicing and sonication of seed for two days as well as late priming by mixture of fungal strains for five days. The outcomes revealed that the amount of glyceollin, glycinol (source of glyceollin) and isoflavonoid were maximum with primed and elicited (reactive oxygen species with fungal mixture) and sonication with fungal mixture dual effect. These data confirmed the importance of fungal mixture, wounding stress and reactive oxygen species on the production of glyceollin and isoflavonoid present in soybean plant (Kalli et al. 2020).

1.4.2.7 Effect of Fungal Biotic Elicitor in Sign-Al Transduction in Potato Tubers

Kawakita et al. experimentally confirmed the positive role of fungal elicitor (Phytophthora infestans) in the involvement of guanosine triphosphate and guanosine triphosphatase activity in the tubers of potato. The experiment was started with reaction between potato tuber and hyphae wall component of P. infestans in presence of sorbitol, potassium metametabisulfate, and salicylhydroxamic acid and phenylmethylsulfonyl fluoride followed by centrifugation at 14,000 rotations per minute for fifteen minutes and again recentrifuged at one hour. The outcomes revealed that relative gamma guanosine triphosphatase activity was increased with 1 micromolar concentration of adenosine triphosphate upto 5 h with P. infestans elicitor. So these outcomes confirmed the importance of fungal strains in signal transduction in potato tuber (Kawakita and Doke 1994).

1.4.2.8 Effect of Carbohydrate on Secondary Metabolite Accumulation in Fagonia indica

Khan et al. scientifically proved the importance of carbohydrates (sucrose, glucose, fructose and maltose) on the accumulation of secondary metabolites (phenolic compounds and chlorophyll) in Fagonia indica plant. The experiment was started with the formation of aforementioned plant callus culture by treating the explant of plant stem portion with different concentrations (1, 3 and 5%) of sucrose, glucose, fructose and maltose in MS medium followed by estimation of callus dimension, maximum production of biomass, total phenolic compound production, chlorophyll and estimation of free radical scavenging activity. The outcomes revealed the maximum width and height of callus were obtained with 5% and 3% sucrose concentration, respectively; maximum biomass production from fresh weight and dry weight were obtained from 3 and 5% sucrose concentration, respectively; maximum phenolic compounds were gained from 5% of maltose; phloroglucinol was the most accumulated phenolic compound obtained from all the carbohydrate callus as well as maximum radical scavenging activity was observed with 3% glucose elicitation (Fig. 1.7). So these data confirmed the importance of different carbohydrates on secondary metabolite accumulation in Fagonia indica (Khan et al. 2018).

Fig. 1.7
figure 7

Copyright permission obtained from Khan et al. @ 2018 Elsevier B.V

Callus cultures as affected by various concentrations of Sucrose (S), Glucose (G), Fructose (F) and Maltose (M) 42 days after culture initiation.

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1.4.2.9 Effect of Dextran on Secondary Metabolite Accumulation and Improve Defense in Tomato Fruit

Lu et al. experimentally proved the importance of dextran on the accumulation of phenylpropanoid and flavonoids along with improved defense mechanism against grey mold infection in tomato (Solanum lycopersicum) fruit. The experiment was started with treatment between sterilized wounded tomato fruit (sodium hypochlorite treated) and dextran (0.1%, 0.5% and 1.0%) followed by ruthenium red treatment on the site of wound to view the effect of calcium channel blockers on site of wound as well as activity against grey mold infection Botrytis cinerea inoculation with dextran elicitor. The checked activities enlisted with percent disease incidence and development of lesion with dextran elicitor alone or along with ruthenium red after one and three days of inoculation on original and synthetic wounds after two days; total phenolic and flavonoid accumulation; phenylalanine lyase activity along with percent grey mold infection germination rate. The outcomes revealed that development of disease incidence and induction lesion were decreased with gradual increase in dextran concentration but the incidence was slightly higher with dextran and ruthenium red combination; accumulation of total phenolic and flavonoid compounds were higher with dextran elicitation after one day and two days of inoculation, respectively; the phenylalanine lyase activity was higher with dextran treatment after twelve hour of inoculation but it hits the lowest point after two of inoculation also the rate of spore germination in presence of B. cinerea was gradually decreased with dextran treatment. So these data confirm the importance of dextran as biotic elicitor to strengthening the plant against grey mold and greater accumulation of phenolic compound as well as flavonoid content in tomato fruit (Lu et al. 2019b).

1.4.2.10 Effect of Yeast on Vincristine and Vinblastine Production in Catharanthus roseus Plant

Maqsood et al. scientifically proved the importance yeast as biotic elicitor on the production of vincristine and vinblastine obtained from protoplast sourced tissues and plant of Catharanthus roseus (commonly known as periwinkle). The experiment was commenced from protoplast culture involving incubation between suspended cells and enzymes treatment (cellulose, pectinase, macroenzyme and driselase) in MS medium followed by treatments with NAA, dichlorophenoxyacetic acid, BA and gibberellin within the same medium to culture the callus and embryo formation followed by elicitation using different concentrations of yeast (0.5, 1.0, 1.5 and 2.0 g/L). The proliferation, maturation and germination of vincristine and vinblastine after different yeast elicitation. The outcomes showed that maximum observations were observed with 1.5 and 2.0 g/L yeast concentrations, so these elicitations were selected for enzymatic activities (catalase, superoxide dismutase, ascorbate peroxidase and glutathione reductase) in the medium with different plant regulators. The results showed that maximum catalase and ascorbate peroxidase activities were observed with leaf harvested tissues with 2.0 g/L yeast in the medium contained BA and NAA; the superoxide dismutase and glutathione peroxidase activities were observed with 2.0 g/L yeast in the medium contained BA and NAA in embryo germination stage (Fig. 1.8). So these outcomes confirmed the importance of yeast on production of vincristine and vinblastine in Catharanthus roseus plant (Maqsood and Mujib 2017).

Fig. 1.8
figure 8

Copyright permission obtained from Maqsood et al. @ 2017 Sociedade Brasileira de Farmacognosia

a Isolated protoplasts; b development of embryogenic callus from protoplasts; c individual embryo developed from PDEC treated with T3 treatment of YE and d regenerated plantlet from protoplast derived embryo, grown in MS medium containing yeast.

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1.4.2.11 Effect of Chitosan on Curcumin Production and Improved Defense in Curcuma longa Plant

Sathiyabama et al. experimentally proved the importance of chitosan polysaccharide on the curcumin production, increased biomass production from leaf and rhizome followed by estimation of protein and enzymatic activities against beta glucanase, peroxidase and polyphenol oxidase enzymes. The experiment was started with the reaction between new root/leaf of turmeric obtained from sodium hypochlorite treated turmeric rhizomes and chitosan (0.1% weight/volume) followed by elicited for a time period of six month. During the period, proper dosing of chitosan elicitation was provided after seven month leaves and roots were evaluated for the activities. The outcomes revealed that rhizome biomass was higher with fresh weight culture; chitosan elicitation was increased the curcumin production followed by elicitated leaves produced higher amount of protein as well as greater enzymatic activities against beta glucanase, peroxidase and polyphenol oxidase enzymes than elicitated rhizome. So these data confirmed the importance of chitosan on the accumulation of curcumin and enzymatic activity of turmeric plant (Sathiyabamaa et al. 2016).

1.4.3 Production of Secondary Metabolites Using Abiotic-Biotic Dual Elicitors

1.4.3.1 Effects of Microbial Elicitors on Glycyrrhizic Acid Production in Taverniera cuneifolia Culture

Awad et al. experimentally proved the importance of microbial elicitors on the production of glycyrrhizic acid from root cultures of Taverniera cuneifolia (Indian liquorice) plant. The experiment was started with the development of root from synthetic roots of the plant followed by treatment with fungal (Aspergillus niger, Aspergillus tenius, Penicillium fellutanum, Fusarium moniliforme, Mucor hiemalis) and bacterial (Bacillus aminovorans, A. rhizogenes, A. tumefacians, B. cereus, Rhizobium leguminosarum) cultures. Another elicitation process include treatment of root cultures (after 6th week) with MJ (1, 2.5, 5, 10, 100 and 1000) μM concentration. After three days treatment both elicited root cultures were stored and measured for glycyrrhizic acid production. The outcomes revealed that maximum glycyrrhizic acid was produced from Fusarium moniliforme and R. leguminosarum microbial elicitors. Another outcomes showed that glycyrrhizic acid was maximum obtained with MJ from plant biomass with 100 μM concentration (Fig. 1.9) These data confirmed the importance of microbial elicitors and MJ on the production of glycyrrhizic acid present in T. cuneifolia plant (Awad et al. 2014).

Fig. 1.9
figure 9

Copyright permission obtained from Awad et al. @ 2014 Elsevier B.V

Root cultures of T. cuneifolia. a Mother culture used for root culture initiation and b six weeks old root culture used for elicitation.

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1.4.3.2 Effects of Yeast and MJ on β-thujaplicin in Cupressus lusitanica Culture

Zhao et al. scientifically proved the importance of yeast as biotic elicitor on the production of natural antimicrobial agent generated from geranyl pyrophosphate present in Cupressus lusitanica cell culture. The process was stared with the formation of fresh suspension culture of the plant from fresh cells (4 g) followed by addition yeast (1 mg/ml) on to the five days old culture. Then in a single treatment all plant growth regulators such as calcium ionophore, cholera toxin, mastopaoran and actinomycin were added alone or along with twenty minutes prior to elicitation. The outcomes revealed that β-thujaplicin was greater in production with yeast elicitor within 2–4 days of incubation. The enzymatic activities against isopentenyl pyrophosphate isomerase, geranyl pyrophosphate synthase and monoterpene synthase showed that maximum activity within 1–2 days incubation with yeast elicitor. In all the cases MJ was just one step down than yeast elicitation but greater than dimethylsulfoxide control. These data confirmed the importance of yeast on the natural antimicrobial β-thujaplicin production from C. lusitanica callus culture (Zhao et al. 2006).

1.4.3.3 Effects of Yeast, Chitosan, MJ and Heat on Secondary Metabolite Accumulation in Khus Root Extracts

Moon et al. scientifically proved the importance of yeast, chitosan, MJ and heat as dual abiotic-biotic elicitor on the production of para hydroxyl benzoic acid, vanillin, para coumaric acid, ferulic acid, total phenolic content followed by antioxidative effects using DPPH (Diphenyl picrylhydrazyl), ABTS (Azino bisethyl benzothiazoline sulfonic acid) and FRAP (Ferric reducing ability of plasma) methods as well as acetycholinesterase activity. The experiment was started with reaction between (5–6) long root of khus plant and chitosan biotic elicitor with 100, 200 and 300 mg/l or MJ with 25, 50 and 75 micromolar concentration or yeast elicitor with 5, 10 and 15 mg/ml concentration with or without treatment with 100 °C for twenty minute period. The outcomes revealed that maximum para hydroxyl benzoic acid, vanillin, para coumaric acid, ferulic acid, total phenolic content were obtained from heat treated chitosan (200 mg/l) concentration elicited khus dry weight. Antioxidative efficiency and acetycholinesterase data depicted that best activity observed with heat treated chitosan (200 mg/l) concentration elicited khus dry weight. So these data confirmed the importance chitosan on the production of secondary metabolite from khus plant (Moon et al. 2020).

1.4.3.4 Effects of Yeast and MJ on Silymarin Production in Silybum marianum Culture

Sanchez-Sampedro et al. experimentally proved the importance of yeast and methyl as dual abiotic-biotic elicitor on the production and accumulation of silymarin obtained from Silybum marianum cell culture. The process was started with development of cell suspension culture from hypocotyl callus of the plant cultured in MS medium containing sucrose, dichlorophenoxyacetic acid, benzyladenine, then the suspension culture was elicited with 1 mg/ml concentration of yeast and 100 micromolar concentration of MJ for a period of 3 days. Also the silymarin production was monitored in presence of calcium antagonist, calcium effectors and inhibitors of protein kinase and protein phosphatase enzymes. The outcomes showed that MJ increased silymarin production many folds than yeast. It was also observed that silymarin production was exponentially increased with ruthenium red, neomycin and diphenylene iodonium treated both MJ and yeast elicitors. These data confirmed the importance of MJ and yeast elicitors on silymarin production present in S. marianum suspension culture (Sanchez-Sampedro et al. 2008).

1.4.3.5 Effects of MJ, Phenylacetic Acid and Light Elicitors on Ajuga bracteosa

Ali et al. scientifically proved the importance of MJ, phenylacetic acid and light as dual abiotic and biotic elicitors on the production of volatile oils (α-phelendrene, Sabinene, α-terpinene, limonene, 1,8-cineole, γ-terpinene, β-pinene, β-myrcene, D-limonene, β-phelendrene, β-ocimene, Thujyl alcohol, cis-sabinol, β-linalool, 1-terpinene-4-ol, Cis-geraniol, α-terpineol, myrtenol, myrtenal, nerol, caryophyllene, β-farnesene, carvone, citronellyl acetate, p-cymen-7-ol, bornyl acetate) from cell cultures of Ajuga bracteosa. The process was started with the reaction between callus culture and plant growth regulators (kinetin, BA, dichlorophenoxyacetic acid, indole butyric acid) and elicitors (MJ and phenyl acetic acid) with 0.5, 1.0, 1.5 g/l of concentrations in three different luminous condition such as dark for one day, 16 h light with 8 h dark and complete light for one day. The amount of dry biomass, total phenolic content, total flavonoid content, free radical scavenging activity as well as volatile oils productions were evaluated. The outcomes showed that amount of dry biomass was maximum (11.3 g/l) with BA (1.0 g/l) in complete dark condition; total phenolic content was maximum (7.0 mg of gallic acid equivalent/g) with MJ (0.5 g/l) in complete light condition; total flavonoid content was maximum (3.8 mg quercetin equivalent/g) with MJ (0.5 g/l) in complete light condition; percent free radical scavenging activity was greater (86%) with MJ (0.5 g/l) in complete light condition; higher amount of volatile oils was observed with MJ (0.5 g/l) and BA (1.0 g/l) in dark period. So these data confirmed the importance of MJ, phenylacetic acid, other plant regulators and light treatment on the production and accumulation of secondary metabolites obtained from A. bracteosa cell culture (Ali et al. 2018).

1.4.3.6 Effects of Salicylic Acid, Yeast and Casein Hydrolysate on Colchicine and Thiocolchicoside Production from Gloriosa superba Plant

Mahendran et al. scientifically proved the importance of salicylic acid, yeast, casein hydrolysate and silver nitrate on the accumulation of colchicine and thiocolchicoside in Gloriosa superba (Calihari) plant. The process was started with reaction between cell suspension culture (generated from plant rhizomes cultured in MS medium contained sucrose, dichlorophenoxyacetic acid, NAA) and salicylic acid (13.812, 27.624, 41.436, 55.248 and 69.060 mg/l), yeast, casein hydrolysate and silver nitrate with (100, 200, 300, 400 and 500 mg/l) followed by estimation of colchicine and thiocolchicoside after fifteen and thirty days of elicitation. The outcomes showed that a proper callus induction was happen with a combination of 2.0 mg/l of dichlorophenoxyacetic acid and 0.5 mg/l of NAA as well as maximum colchicine was obtained from casein hydrolysate (300 mg) and (27.624 mg) of salicylic acid after fifteen and thirty days of elicitation respectively whereas maximum thiocolchicoside was obtained from silver nitrate (200 mg) and (300 mg) of silver nitrate after fifteen and thirty days of elicitation respectively. So these data confirmed the importance of mixed abiotic and biotic elicitors on the accumulation of colchicine and thiocolchicoside in G. superba plant (Mahendran et al. 2018).

1.4.3.7 Effects of Yeast and MJ on Polyphenolic Compound in Aster scaber Plant

Ghimire et al. scientifically proved the importance of biotic elicitor yeast and abiotic elicitor MJ on the production of polyphenolic compounds (flavonols, hydroxy cinnamic acid derivative, hydroxybenzoic acid derivative, vanillin, resveratrol and homogentisic), biomass production, total phenolic and flavonoid content as well as effects on antioxidative properties of the hairy root culture of the plant using diphenylpicraylhydrazyl and ferric reducing potential processes. The experiment was started with the formation of hairy root culture of A. scaber plant (Chwinamul) by the germination of seeds in MS medium contained with sucrose, cefotaxime antibiotic and excised with A. rhizogenes followed by elicited with yeast with 50 mg/l, 100 mg/l, 200 mg/l concentration and MJ with 50, 100, 200 micromolar concentration. The outcomes revealed that maximum biomass was obtained with MS medium, Sucrose (3%) with twenty seven days of interval. Maximum flavonol, hydroxy cinnamic acid derivative, hydroxybenzoic acid derivative, vanillin, resveratrol and homogentisic were obtained with MJ elicitation (Fig. 1.10). Total phenolic and flavonoid contents were higher with increased dose of both elicitors as well as MJ (100 micromolar concentration) was showed maximum antioxidative property. So these data confirmed the importance of MJ and yeast as elicitors on the accumulation of polyphenols in A. scaber plant (Ghimire et al. 2019).

Fig. 1.10
figure 10

Copyright permission obtained from Ghimire et al. @ 2019 Elsevier B.V

Agrobacterium rhizogenes-mediated hairy root cultures in Aster scaber. a Hairy roots induction, b Hairy root cultures in hormone-freeMS liquid medium, c PCR analysis of the rolC gene in the transgenic root lines. DNA ladder marker Lane M, pRiKCTC2703 DNA C(+), transgenic root lines induced by A. rhizogenes L1–L5, roots from a non-transgenic plant C(−).

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1.4.3.8 Effects of Phenylalanine, Salicylic Acid and Chitosan on Secondary Metabolites of Coleus aromaticus Benth

Govindaraju et al. experimentally confirmed the importance of phenylalanine, salicylic acid and chitosan as dual abiotic-biotic elicitor on the accumulation of alkaloid, flavonoid, saponin, terpenoids, total phenolic content followed by expression of phenylalanine messenger ribonucleic acid in the root/shoot culture and regenerated Coleus aromaticus Benth plant. The experiment was started from the induction of shoot followed by development of explants in the MS medium with activated charcoal and ascorbic acid as carbon and antioxidant source. The effect of BA and kinetin were also tested. The outcomes revealed that healthy explants were maximized with (7.5 g/l) activated charcoal and (1.0 mg/l) ascorbic acid. The percent induction of explants and number of shoots were maximum with a combination of (1 mg/l) of BA and kinetin. Different concentration of phenylalanine (0.5, 1.0, 1.5, 2.0, and 2.5) mg/l, salicylic acid (0.2, 0.4, 0.6, 0.8, and 1.0) mg/l and chitosan (20, 40, 60, and 80) mg/l were used as elicitors with BA (1 mg/l) concentration. The outcomes showed that maximum number of shoots were observed with 40 mg/l of chitosan cultured with BA as well as maximum development of roots was observed with (0.5 mg/l) of a combination NAA and IAA (Fig. 1.11). The outcomes also showed that regenerated explants observed with greater amounts of alkaloid, flavonoid, saponin, terpenoids, total phenolic content as well as higher expression of phenylalanine messenger ribonucleic acid. These data confirmed the effects of phenylalanine, salicylic acid and chitosan on secondary metabolites of C. aromaticus Benth (Govindaraju and Arulselvi 2018).

Fig. 1.11
figure 11

Copyright permission obtained from Govindaraju et al. @ 2016 Elsevier B.V

In vitro propagation of C. aromaticus, a shoot bud induction, b multiple shoot bud development from in vitro derived nodal explants, c multiple shoots (1.0 mg BAP + 40 mg/l Ch), d root initiation and e, f rooted plant growing in the plastic cup with soil and sand in the ratio of 1:1.

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1.4.3.9 Effects of MJ, Chitosan and Microbial Lysates on Essential Oil Accumulation in Rhododendron tomentosum

Jesionek et al. experimentally proved the importance of abiotic elicitors (copper, nickel, methyl jasmoante in dimethyl sulfoxide and ethanol, dimethylsulfoxide, ethanol) and biotic elicitors (chitosan hydrochloride, ergosterol and aphid extract in ethanol, lysates of C. albicans, E. coli, Enterobacter sakazaki, Pectobacterium Carotovorum, Dickeya dadantii) on the quantity of essential oils (monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, aromadendrane-type sesquiterpenes) in R. tomentosum plant. The experiment was started with formation of in vitro culture of the plant on Schenk-Hildebrandt medium contained ammonium nitrate and isopentenyladenine within a period of one month. The formation of biomass was done using microshoots of the plant within sterilized bioreactor with proper illumination followed by elicitation using the aforementioned abiotic and biotic elicitors. The fresh weight and dry weight of biomass with growth index reflected that nickel, MJ in ethanol, ergosterol in ethanol and lysate of Enterobacter sakazaki showed greater production. The productions of monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpene and aromadendrane-type sesquiterpenes were maximized with copper and moderate with Escherichia coli lysates, nickel and ethanolic ergosterol and ethanolic MJ, respectively. The outcomes stated the importance of both abiotic and biotic elicitors on the production of volatile oils in R. tomentosum plant (Jesionek et al. 2018).

1.4.3.10 Effects of Yeast, MJ and Salicylic Acid on Ursolic Acid and Eugenol Production in Ocimum tenuiflorum L.

Sharan et al. experimentally proved the importance of abiotic elicitors [(MJ: 0, 3, 60 and 120 mg/l) and (salicylic acid: 0, 30, 60 and 120 mg/l)] and biotic elicitor (yeast: 0, 25, 50 and 100 mg/l) on the production of ursolic acid and eugenol in Ocimum tenuiflorum L (holy basil) plant. The experiment was started with formation of hairy root culture from explants cultured in MS medium with cefotaxime and sucrose as antibiotic and sugar source, respectively followed by elicitation using aforementioned elicitors and evaluated the effects of A. rhizogenes bacterial strains (LBA 9402 and A4) on the transformation into leaf and stems as well as evaluated the production of biomass, ursolic acid and eugenol contents for a time period of thirty five days and finally quantified the effects of elicitors on seventeen and twenty one old hairy roots tendency to accumulate ursolic acid and eugenol within four to twelve days period. The outcomes revealed that LBA 9402 strain of A. rhizogenes facilitated the transformation of explants into leaf and stem; highest amounts of biomass, ursolic acid and eugenol accumulations were observed within twenty to twenty five days of incubation as well as maximum ursolic acid was accumulated with (50 mg/l) yeast, (60 mg/l) of methyl jasmoante and (60 mg/l) of salicylic acid from seventeen day old hairy roots and (50 mg/l) yeast, (0 mg/l) of methyl jasmoante and (0 mg/l) of salicylic acid from twenty one day old hairy roots; whereas maximum eugenol was accumulated with (50 mg/l) yeast, (60 mg/l) of methyl jasmoante and (0 mg/l) of salicylic acid from seventeen day old hairy roots and (0 mg/l) yeast, (0 mg/l) of methyl jasmoante and (0 mg/l) of salicylic acid from twenty one day old hairy roots. These data confirmed the importance of yeast, MJ and salicylic acid contents present in O. tenuiflorum L plant (Sharan et al. 2019).

1.5 Conclusion

This chapter provides a detailed information about What is Elicitation? Types of Elicitors along with applications of elicitors to increase productivity of secondary metabolites. This chapter also includes the information about secondary metabolites and its types such as alkaloid, glycoside, flavonoid and terpenoids etc. Elicitors are the physical/chemical/biological/microbial substances which under stress conditions induce the biosynthesis of secondary metabolites of plants. Both biotic and abiotic elicitors are used in the process. Most common secondary metabolites Ferulic acid, cinnamic acid, vanillin, coumaric acid, silymarin, affinin, hypocrellin A, steroiside, menthone, piperitone, glycyrrhizic acid, colchicine, thiocolchicoside, phenolic acid, gymnemic acid, flavonoids are utilized the elicitation technique. Elicitors are two types such as: abiotic and biotic. Abiotic elicitors such as salicylic acid, methyl jasmonate, hydrogen peroxide, lanthanum, different hormones, light, gamma rays and controlled temperature are used to generate secondary metabolites of wheat grass, Thymus vulgaris, Silybum marianum, Shiraia bambusicola, Ajuga bracteosa, broccoli plant, etc. Biotic elicitors like chitosan, rhizobacteria, Rhizobium leguminosum, A. tenius, A. tumefacians, carrageenan, Streptomyces, Rhizopus, dextran, yeast are used to develop or improvise secondary metabolites of Khus, M. pulegium, T. cuneifolia, chickpea, V. vinifera, R. gmelini Turcz, C. lusitanica, etc. Some secondary metabolites of C. aromaticus Benth, R. tomentosum, F. indica, R. serpentine, S. khasianum, O. tenuiflorum, S. rebaudiana etc. are used both abiotic and biotic elicitors. In the Table 1.1 detailed tabulated information of elicitors used to accumulate and increased productivity of secondary metabolites. Elicitors are not only increase secondary metabolite production as well as increase the defense mechanism, antioxidative, antimicrobial and enzymatic activities. This data collectively express the detailed knowledge about the elicitors and its signal transduction behavior. So if scientific minds hunts for secondary metabolites with greater activity but with very lesser abundance, then elicitation is the future.

Table 1.1 Detailed effects of elicitors on different plants and secondary metabolites
Full size table