Abiotic and Biotic Elicitors-Role in Secondary Metabolites Production Through in Vitro Culture of Medicinal Plants

Chapter 10
Abiotic and Biotic Elicitors–Role in Secondary

Metabolites Production through In Vitro Culture of
Medicinal Plants
Poornananda M. Naik and Jameel M. Al–Khayri
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/61442
Abstract
Plant secondary metabolites are having the great application in human health and nutri‐
tional aspect. Plant cell and organ culture systems are feasible option for the production
of secondary metabolites that are of commercial importance in pharmaceuticals, food ad‐
ditives, flavors, and other industrial materials. The stress, including various elicitors or
signal molecules, often induces the secondary metabolite production in the plant tissue
culture system. The recent developments in elicitation of plant tissue culture have opened
a new avenue for the production of secondary metabolite compounds. Secondary metab‐
olite synthesis and accumulation in cell and organ cultures can be triggered by the appli‐
cation of elicitors to the culture medium. Elicitors are the chemical compounds from
abiotic and biotic sources that can stimulate stress responses in plants, leading to the en‐
hanced synthesis and accumulation of secondary metabolites or the induction of novel
secondary metabolites. Elicitor type, dose, and treatment schedule are major factors de‐
termining the effects on the secondary metabolite production. The number of parameters,
such as elicitor concentrations, duration of exposure, cell line, nutrient composition, and
age or stage of the culture, is also important factors influencing the successful production
of biomass and secondary metabolite accumulation. This chapter reviews the various
abiotic and biotic elicitors applied to cultural system and their stimulating effects on the
accumulation of secondary metabolites.
Keywords: Cell culture, elicitor, organ culture, secondary metabolites, stress
1. Introduction
The total mankind is dependent on plants as a source of carbohydrates, proteins, vitamins,

food, and shelter. Plants are studied for their important constituents and the nutritional factors
© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
248 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives
for over decades. Along with the essential primary metabolites, higher plants are also capable
to produce a number of low molecular weight compounds. A diverse group of organic
compounds that are produced by plants to facilitate interaction with the biotic environment
and the establishment of a defense mechanism are called as plant secondary metabolites [1–
3]. The production of these metabolites is very low (less than 1% dry weight) and depends
greatly on the physiological and developmental stage of the plant [4,5]. Plant natural products
have been an important part of medicine throughout human history. In recent years, the use
of herbal medicines has steadily increased worldwide [6]. With this increasing demand comes
growing concerns about the safety and efficacy of herbal medicines. Although the potential
for medicinal plants seems almost limitless, there are a few major obstacles that hinder large-
scale utilization by the western medical system. Among them is the lack of reproducibility
common in testing many plant extracts (up to 40%), which has limited the enthusiasm for
developing plant-based pharmaceuticals [7]. Unlike standardized single-entity pharmaceuti‐
cal drugs, herbal medicines consist of complex mixtures with multiple compounds responsible
for therapeutic activity, making standardization difficult [8]. Further complicating the issue is
the fact that plants, unlike synthetic medicines, are living organisms, with inherent biological
variation [9]. Just because plant material originates from the same species, it does not neces‐
sarily mean that the chemical content will be identical. This lack of reproducibility may be due
to two main factors, genetic variability and differences in growing conditions.
In addition, plants are a valuable source of a wide range of secondary metabolites, which are
used as pharmaceuticals, agrochemicals, flavors, fragrances, colors, biopesticides, and food
additives. Plants are producing new compounds and in future new chemical models are
drawing for new drugs because the most of the plants chemistry is yet to be explored [10]. The
characterization of molecular structures and chemical analysis helped us to pinpoint the
activities of plants under controlled conditions. Although all these advancements, we still
depend on the secondary metabolites of biological sources including pharmaceuticals [11].
Due to various agro alimentary, perfumes, flavors, colors, and pharmacological effects, the
secondary metabolites are having extensive demand and various commercial preparations are
available in the market. Besides, the appeal of using natural products for medicinal purposes
is increasing, and metabolic engineering can alter the production of pharmaceuticals and help
to design new therapies. The evolving commercial importance of secondary metabolites has
in recent years resulted in a greater interest in secondary metabolism, particularly in the
possibility of altering the production of bioactive plant metabolites [12]. Secondary metabolites
are separated into nitrogen compounds (alkaloids, nonprotein amino acids, amines, alcamides,
cyanogenic glycosides, and glucosinolates) and nonnitrogen compounds (monoterpenes,
diterpenes, triterpenes, tetraterpenes, sesquiterpenes, saponins, flavonoids, steroids, and
coumarins).
The plant tissue culture plays an important role in the rapid clonal propagation, regeneration
of genetically manipulated superior clones, conservation of germplasm, production of
secondary metabolites, and ex vitro conservation of valuable phytodiversity [13,14]. The plant,
Abiotic and Biotic Elicitors: Role in Secondary Metabolites Production through... 249
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cell, tissue and organ culture techniques have come up with an escapable tool with the
possibilities of acclaiming and supplementing the conventional method in plant breeding,
plant improvement, and biosynthetic pathways. This technique has several potential applica‐
tions in crop improvement, and efficient regeneration is a prerequisite in such improvement
programs. The biotechnological production of secondary metabolites in plant cell and organ
cultures is an attractive alternative to the extraction of the whole plant material [15]. In
particular, plant–specific important compounds are obtained by using the plant cell and organ
cultures [2]. The faster proliferation rates and shorter biosynthetic cycle of cell and organ
cultures leads to have a higher rate of metabolism when compared to field grown plants [16].
Further, plant cell/organ cultures are under controlled conditions proliferates at their optimum
growth rates when compared to the cultivated plants, which are facing environmental,
ecological, and climatic variations. In recent years, various strategies have been developed for
use in biomass accumulation and the synthesis of secondary compounds, such as strain
improvement, optimization of medium, and culture environments, elicitation, precursor
feeding, metabolic engineering, permeabilization, immobilization, and biotransformation
methods, bioreactor cultures, and micropropagation [17]. The focus of the present chapter is
the influence of abiotic and biotic elicitors on the secondary metabolite production in the in
vitro cultured medicinal plants.
2. Classification of elicitors and secondary metabolite production via in

vitro culture of medicinal plants
Stress is an important factor in determining the chemical composition and therapeutic activity
of medicinal plants. Actively stimulating, or eliciting, the plant stress response to induce the
desired chemical response is called elicitation, harnessing the connection between plant stress
and phytochemistry. “Elicitor may be defined as a substance for stress factors which, when
applied in small quantity to a living system, it induces or improves the biosynthesis of specific
compound which do have an important role in the adaptations of plants to a stressful condi‐
tions” [18]. Elicitation is the induced or enhanced biosynthesis of metabolites due to addition
of trace amounts of elicitors [18]. Several biotechnological strategies have been hypothesized
and applied for the productivity enhancement, and elicitation is recognized as the most
practically feasible strategy for increasing the production of desirable secondary compounds
from cell, organ, and plant systems [19–21].
On the basis of nature, elicitors can be divided into two types abiotic and biotic (Figure 1).
Abiotic elicitors comprise of substances that are of nonbiological origin and are grouped in
physical, chemical, and hormonal factors. Biotic elicitors are the substances of biological origin
that include polysaccharides originated from plant cell walls (e.g. chitin, pectin, and cellulose)
and micro–organisms.
Figure 1. Elicitors Classification Based on their Nature
3. Abiotic elicitors
As mentioned above, the abiotic elicitors are categorized into physical, chemical, and hormonal
elicitors. Abiotic elicitors have wide range of effects on the plants and in the production of
secondary metabolites (Table 1).
Elicitor Plant species Nature of culture Compounds References
Ozone (O3) Melissa officinalis Shoot Rosmarinic acid [163]
Hypericum perforatum Cell suspension Hypericin [164]
Pueraria thomsnii Cell suspension Puerarin [165]
pH Bacopa monnieri Shoot Bacoside A [166]
Withania somnifera Hairy root Withanolide A [167]
Withania somnifera Cell suspension Withanolide A [168]
Sucrose Hypericum adenotrichum Seedling Hypericin and [41]

pseudohypericin
Corylus avellana Cell suspension Paclitaxel [169]
Bacopa monnieri Shoot Bacoside A [166]
Withania somnifera Cell suspension Withanolide A [168]
Ultraviolet C Vitis vinifera Cell suspension Stilbene [34]
Proline Stevia rebaudiana Callus and suspension Steviol glycoside [40]
Polyethylene glycolStevia rebaudiana Callus and suspension Steviol glycoside [40]

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Hypericum adenotrichum Seedling Hypericin and [41]

pseudohypericin
Jasmonic acid Bacopa monnieri Shoot Bacoside A [170]
Plumbago indica Hairy root Plumbagin [88]
Plumbago rosea Cell suspension Plumbagin [86]
Methyl jasmonate Salvia miltiorrhiza Hairy root Tanshinone [94]
Perovskia abrotanoides Adventitious roots Cryptotanshinone and [78]

tanshinone IIA
Vitis vinifera Cell suspension Stilbene [34]
Bacopa monnieri Shoot Bacoside [96]
Salvia officinalis Shoot Diterpenoid [171]
Silybum marianum Cell suspension Silymarin [172]
Salvia castanea Hairy root Tanshinone [173]
Gymnema sylvestre Cell suspension Gymnemic acid [148]
Withania somnifera Hairy root Withanolide A, [95]

withanone, and
withaferin A
Andrographis paniculata Cell suspension Andrographolide [97]
Vitis vinifera Cell suspension trans-Resveratrol [91]
Taverniera cuneifolia Root Glycyrrhizic acid [135]
Gibberelic acid Salvia miltiorrhiza Hairy root Tanshinones [174]
Echinacea pupurea Hairy root Caffeic acid derivatives [175]
Salicylic acid Salvia miltiorrhiza Hairy root Tanshinone [94]
Vitis vinifera Cell suspension Stilbene [34]
Digitalis purpurea Shoot Digitoxin [176]
Hypericum hirsutum Shoot Hypericin and [177]

pseudohypericin
Gymnema sylvestre Cell suspension Gymnemic acid [148]
Withania somnifera Hairy root Withanolide A, [95]

withanone, and
withaferin A
Datura metel Root Hyoscyamine and [178]

scopolamine
Glycyrrhiza uralensis Adventitious root Glycyrrhizic acid [179]

Sodium salicylate Salvia officinalis Shoot Carnosol [180]
Sodium chloride Catharanthus roseus Embryogenic tissues Vinblastine and [55]

vincristine
Sorbitol Perovskia abrotanoides Adventitious roots Cryptotanshinone and [78]

tanshinone IIA
Silver (Ag) Perovskia abrotanoides Adventitious roots Cryptotanshinone and [78]

tanshinone IIA
Vitis vinifera Cell suspension Resveratrol [67]
Salvia castanea Hairy root Tanshinone [173]
Datura metel Hairy root Atropine [150]
Cadmium (Cd) Vitis vinifera Cell suspension Resveratrol [67]
Datura stramonium Root Sesquiterpenoid [79]
Cobalt (Co) Vitis vinifera Cell suspension Resveratrol [67]
Copper (Cu) Ammi majus Shoot Xanthotoxin [181]
Bacopa monnieri Shoot Bacoside [170]
Datura stramonium Root Sesquiterpenoid [79]
Table 1. Effect of Different Abiotic Elicitors on the Production of Various Secondary Metabolites in Plants
3.1. Physical elicitors
Physical elicitors include light, osmotic stress, salinity, drought, and thermal stress.
3.1.1. Light
The light is a physical factor that can affect the metabolite production. Light can stimulate such
secondary metabolites include gingerol and zingiberene production in Zingiber officinale callus
culture [22]. The effect of light irradiation on anthocyanin production in cell suspension
cultures of Perilla frutescens was reported [23]. The effect of light and hormones on the digitoxin
accumulation in Digitalis purpurea L. was reported by Hagimori et al. [24]. Moreover, in hairy
root cultures of Artemisia annua, the effect of light irradiation influenced the artemisinin
biosynthesis [25]. The effect of white light on taxol and baccatin III accumulation in cell cultures
of Taxus cuspidate was reported by Fett–Neto et al. [26]. Ultraviolet (UV) radiation stimulates
secondary metabolite production. Increasing UV–B exposure in field–grown plants not only
increased the total essential oil and phenolic content but also decreased the amount of the
possibly toxic beta–asarone [27]. These findings are to be expected as phenolics are known UV
protectants [28]. Catharanthus roseus plants, exposed to UV–B light, show significant increases
in the production of vinblastine and vincristine, which have proven effective in the treatment
of leukemia and lymphoma [29]. UV–C irradiation promotes the phenylpropanoid pathway
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and stimulates flavonoid synthesis [30]. UV–C irradiation is an effective method to enhance
stilbene production in Vitis vinifera berries [31], V. vinifera leaves [32], and V. vinifera callus of
different genotypes [33]. UV–C together with methyl jasmonate (MeJA) or salicylic acid (SA)
also used to enhance stilbene production in V. vinifera cell cultures [34].
3.1.2. Osmotic stress
Osmotic stress (water stress) is an abiotic physical elicitor [35] and is one of the important
environmental stresses that can alter the physiological and biochemical properties of plants
and increase the concentration of secondary metabolites in plant tissues [36]. Proline acts as
an osmolyte, as protective agent for cytoplasmic enzymes, as a reservoir of nitrogen and carbon
sources for post–stress growth, or even as a stabilizer of the machinery for protein synthesis,
regulation of cytosolic acidity and scavenging of free radicals [37]. However, the various roles
of proline have been proposed, but the main role could be the osmotic adjustment in osmoti‐
cally stressed plant tissues and the protection of plasma membrane integrity [38]. Polyethylene
glycol (PEG) is an osmotic agent (nonpenetrating osmoticum) that has been used for induction
of water stress in many plants [39]. The proline and PEG enhanced the production of steviol
glycosides content in both callus as well as suspension culture of Stevia rebaudiana [40]. PEG
elicited the pharmacologically active compounds, such as hypericin and pseudohypericin, in
Hypericum adenotrichum [41]. Sucrose is a typical osmotic stress agent used for the induction
of water stress in plants that also serves as a vital carbon and energy source [42]. It has been
shown that water and osmotic imbalance can strongly influence the synthesis of hypericin and
hyperforin in Hypericum perforatum plants [43]. In addition, it has been reported that both
hypericin and pseudohypericin concentrations decreased, while hyperforin concentration
increased significantly in the plants grown under water stress conditions [36].
3.1.3. Salinity
Salinity reduces plant growth and development and alters a wide array of physiological and
metabolic processes [44,45]. Plants have developed complex mechanisms for adaptation to the
osmotic, ionic, and oxidative stresses that are induced by the salt stress. Exposure to salinity
is known to induce or stimulate the production of secondary plant products, such as phenols,
terpenes, and alkaloids [46–48]. C. roseus grown under salt stress showed increased levels of
the alkaloid vincristine [49]. In Grevillea, a significant increase in anthocyanin concentration
was reported under salinity exposure in both the salt–tolerant Grevillea ilicifolia and the salt–
sensitive Grevillea arenaria [50]. In contrast to this, salt stress decreased the anthocyanin level
in the salt–sensitive species [51]. In Datura innoxia, salt treatment increased the total alkaloid
content in young leaves, and the results indicated that at the organ level, tropane alkaloid
accumulation was related to plant growth [52]. Glycine betaine was increased under salinity
in numerous species including Triticum aestivum [53] and Trifolium repens [54]. Salinity also
increased the diamine and polyamine content in Oryza sativa [53]. An improved synthesis of
vinblastine and vincristine was observed in C. roseus embryogenic tissue culture by using NaCl
as an elicitor [55].
3.1.4. Drought stress
One of the most important abiotic stress is drought, which affect plant growth and their
developmental process [56]. The available water in the soil is reduced to such critical levels,
and atmospheric conditions add to the continuous loss of water; the situation is called drought
stress. The high temperature in the environment and solar radiations add up the water deficit
in the soil, which leads to drought stress. Drought stress tolerance is observed in all types of
plants, but its extent varies from species to species [56]. Drought stress, which can also greatly
reduce plant growth, can increase secondary metabolite content. Mild water stress significantly
increased the content of the anti–inflammatory saikosaponins in Bupleurum chinense [57].
Moderate water stress increased the content of salvianolic acid in roots of Salvia miltiorrhiza,
although the content of other bioactives, including tanshinone, was lowered [58]. Moderate
drought stress also increased the production of rosmarinic, ursolic, and oleanolic acid in
Prunella vulgaris [59]. A weak water deficit greatly increased the glycyrrhizic acid content in
roots of Glycyrrhiza uralensis [60]. In Hypericum brasiliense, the amounts of various phenols and
betulinic acid were drastically increased under drought stress [61].
3.1.5. Thermal stress
Although thermal stress can greatly reduce plant growth and induce senescence, elevated
temperatures (heat stress) or low temperatures (cold stress) have also been shown to increase
secondary metabolite production. Temperature strongly influences metabolic activity and
plant ontology, and high temperatures can induce premature leaf senescence [62]. Elevated
temperatures increase leaf senescence and root secondary metabolite concentrations in the
herb Panax quinquefolius [63]. A 5°C increase in temperature significantly increased the
ginsenoside content in roots of P. quinquefolius [63]. A temperature variation has multiple
effects on the metabolic regulation, permeability, and rate of intracellular reactions in plant
cell cultures [62]. Temperature range of 17–25°C is normally used for the induction of callus
tissues and growth of cultured cells [16]. The temperature and light quality influences on the
production of ginsenoside in hairy root culture of Panax ginseng [64]. The Melastoma malaba‐
thricum cell cultures incubated at a lower temperature range (20 ± 2°C) grew better and had
higher anthocyanin production than those grown at 26 ± 2°C and 29 ± 2°C [65]. Fifteen days at
35°C significantly increased the hypericin and hyperforin content in shoots of Hypericum
perforatum [66].
4. Chemical elicitors
Heavy metals have become one of the main abiotic stress agents for living organisms
because of their increasing use in the developing fields of industry and agrotechnics and
high bioaccumulation and toxicity [67]. Although a lot of information is available concern‐
ing the effects of heavy metals on plant growth and physiology, much less is known
regarding their effects on the production of secondary metabolites. Heavy metal–induced
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changes in metabolic activity of plants can affect the production of photosynthetic pig‐
ments, sugars, proteins, and nonprotein thiols. These effects can result from the inhibition
of enzymes involved in the production of these natural products, likely through impaired
substrate utilization [68]. Metals may alter the production of bioactive compounds by
changing aspects of secondary metabolism [2]. Metals including Ni, Ag, Fe, and Co have
been shown to elicit the production of secondary metabolites in a variety of plants [69].
An increased oil content up to 35% in Brassica juncea was seen due to the effective
accumulation of metals (Cr, Fe, Zn, and Mn) [70]. The highest accumulations of secon‐
dary metabolites such as shikonin [71] and also the production of digitalin [72] were
observed by treating Cu2+ and Cd2+. The production of betalains in Beta vulgaris also
stimulated by Cu2+ [73]. Co2+ and Cu2+ have a stimulatory effect on the production of
secondary metabolites in Beta vulgaris [73]. The betalaines production was enhanced by
exposing the hairy root culture to metal ions [74]. The stimulatory effects of Cu2+ on the
accumulation of betacyanins in callus cultures of Amaranthus caudatus were reported by
Obrenovic [75]. The addition of Zn2+ (900 μM) improved the yield of lepidine in cultures
of Lepidium sativum [76]. However, Cu proved more effective than Zn in enhancing the yield
product [76]. In hairy root cultures of Brugmansia candida, silver nitrate (AgNO3) or cadmium
chloride (CdCl2) elicited the overproduction of two tropane alkaloids, scopolamine, and
hyoscyamine [20]. The production of taxol in cell culture of Taxus sp. was enhanced by the
rare–earth metal (lanthanum) [77]. AgNO3 stimulated the production of tanshinone in the
root culture of Perovskia abrotanoides [78]. The treatment of root cultures of Datura stramoni‐
um with cadmium salts at external concentrations of approximately l mM has been found
to induce the rapid accumulation of high levels of sesquiterpenoid–defensive compounds,
notably lubimin and 3–hydroxylubimin, but not alkaloid [79].
5. Hormonal elicitors
Various plant hormones have been extensively used in elicitation studies. The most studied,
because of their key roles in the plant defense response, are jasmonic acid (JA) and SA and its
derivatives.
5.1. Jasmonates
Jasmonates, including JA and MeJA, are a family of cyclopentanone compounds that modulate
a wide range of plant responses [80,81] and act as effective elicitors to enhance secondary
metabolites in in vitro cultures. They constitute an important class of elicitors for many plant
secondary metabolic pathways, which are typically manifested by the elicitation of secondary
metabolite biosynthesis when plants face particular environmental stresses [82]. JA is an
important signal molecule of plant in response to wound and pathogen attack [83]. JA and its
more active derivative MeJA can induce the production of a wide range of plant secondary
metabolites such as rosmarinic acid, terpenoid indole alkaloid, and plumbagin in various cell
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
production of the raspberry ketone benzalacetone [100].
5.2. Salicylic acid
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].
5.3. Gibberellic acid
Gibberellin (GA), a phytohormone, is also well known as an effective elicitor for the production
of secondary metabolites [109].
6. Biotic elicitors
In the production of secondary metabolites from plants, the use of biotic elicitors had an
important role (Table 2).
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Chitin Hypericum perforatum Shoot Hypericin and [182]

pseudohypericin
Hypericum perforatum Cell suspension Phenylpropanoid and [183]

naphtodianthrone
Vitis vinifera Cell suspension trans-Resveratrol and [91]

viniferins
Pectin Hypericum perforatum Shoot Hypericin and [182]

pseudohypericin
Dextran Hypericum perforatum Shoot Hypericin and [182]

pseudohypericin
Yeast extract Perovskia abrotanoides Adventitious roots Cryptotanshinone and [78]

tanshinone IIA
Plumbago rosea Cell suspension Plumbagin [86]
Silybum marianum Cell suspension Silymarin [172]
Trichoderma atroviride Salvia miltiorrhiza Hairy root Tanshinone [184]
Protomyces gravidus Ambrosia artemisiifolia Hairy root Thiarubrine A [134]
Claviceps purpurea Azadirachta indica Hairy root Azadirachtin [136]
Mucor hiemalis Taverniera cuneifolia Root Glycyrrhizic acid [135]
Fusarium oxysporum Hypericum perforatum Cell suspension Phenylpropanoid and [183]

naphtodianthrone
Phoma exigua Hypericum perforatum Cell suspension Phenylpropanoid and [183]

naphtodianthrone
Botrytis cinerea Hypericum perforatum Cell suspension Phenylpropanoid and [183]

naphtodianthrone
Aspergillus niger Gymnema sylvestre Cell suspension Gymnemic acid [149]
Saccharomyces cerevisiae Gymnema sylvestre Cell suspension Gymnemic acid [149]
Agrobacterium Gymnema sylvestre Cell suspension Gymnemic acid [149]

rhizogenes
Bacillus subtilis Gymnema sylvestre Cell suspension Gymnemic acid [149]
Escherichia coli Gymnema sylvestre Cell suspension Gymnemic acid [149]
Datura metel Hairy root Atropine [150]
Bacillus cereus Datura metel Hairy root Atropine [150]
Staphylococcus aureus Datura metel Hairy root Atropine [150]
Rhizobium Taverniera cuneifolia Root Glycyrrhizic acid [135]

leguminosarum
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
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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
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].
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
http://dx.doi.org/10.5772/61442
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
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.
http://dx.doi.org/10.5772/61442
Author details
Poornananda M. Naik and Jameel M. Al–Khayri*
*Address all correspondence to: [email protected]; [email protected]
Department of Agricultural Biotechnology, College of Agriculture and Food Sciences, King

Faisal University, Al–Hassa, Saudi Arabia
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Abiotic and Biotic Elicitors-Role in Secondary Metabolites Production Through in Vitro Culture of Medicinal Plants

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Chapter 10

Abiotic and Biotic Elicitors–Role in Secondary

Poornananda M. Naik and Jameel M. Al–Khayri

Additional information is available at the end of the chapter

Keywords: Cell culture, elicitor, organ culture, secondary metabolites, stress

The total mankind is dependent on plants as a source of carbohydrates, proteins, vitamins,

2. Classification of elicitors and secondary metabolite production via in

Figure 1. Elicitors Classification Based on their Nature

Elicitor Plant species Nature of culture Compounds References

Ozone (O3) Melissa officinalis Shoot Rosmarinic acid [163]

Hypericum perforatum Cell suspension Hypericin [164]

Pueraria thomsnii Cell suspension Puerarin [165]

pH Bacopa monnieri Shoot Bacoside A [166]

Withania somnifera Hairy root Withanolide A [167]

Withania somnifera Cell suspension Withanolide A [168]

Sucrose Hypericum adenotrichum Seedling Hypericin and [41]

Corylus avellana Cell suspension Paclitaxel [169]

Bacopa monnieri Shoot Bacoside A [166]

Withania somnifera Cell suspension Withanolide A [168]

Ultraviolet C Vitis vinifera Cell suspension Stilbene [34]

Proline Stevia rebaudiana Callus and suspension Steviol glycoside [40]

Polyethylene glycolStevia rebaudiana Callus and suspension Steviol glycoside [40]

Elicitor Plant species Nature of culture Compounds References

Hypericum adenotrichum Seedling Hypericin and [41]

Jasmonic acid Bacopa monnieri Shoot Bacoside A [170]

Plumbago indica Hairy root Plumbagin [88]

Plumbago rosea Cell suspension Plumbagin [86]

Methyl jasmonate Salvia miltiorrhiza Hairy root Tanshinone [94]

Perovskia abrotanoides Adventitious roots Cryptotanshinone and [78]

Vitis vinifera Cell suspension Stilbene [34]

Bacopa monnieri Shoot Bacoside [96]

Salvia officinalis Shoot Diterpenoid [171]

Silybum marianum Cell suspension Silymarin [172]

Salvia castanea Hairy root Tanshinone [173]

Gymnema sylvestre Cell suspension Gymnemic acid [148]

Withania somnifera Hairy root Withanolide A, [95]

Andrographis paniculata Cell suspension Andrographolide [97]

Vitis vinifera Cell suspension trans-Resveratrol [91]

Taverniera cuneifolia Root Glycyrrhizic acid [135]

Gibberelic acid Salvia miltiorrhiza Hairy root Tanshinones [174]

Echinacea pupurea Hairy root Caffeic acid derivatives [175]

Salicylic acid Salvia miltiorrhiza Hairy root Tanshinone [94]

Vitis vinifera Cell suspension Stilbene [34]

Digitalis purpurea Shoot Digitoxin [176]

Hypericum hirsutum Shoot Hypericin and [177]

Gymnema sylvestre Cell suspension Gymnemic acid [148]

Withania somnifera Hairy root Withanolide A, [95]

Datura metel Root Hyoscyamine and [178]

Glycyrrhiza uralensis Adventitious root Glycyrrhizic acid [179]

Elicitor Plant species Nature of culture Compounds References

Sodium salicylate Salvia officinalis Shoot Carnosol [180]

Sodium chloride Catharanthus roseus Embryogenic tissues Vinblastine and [55]

Sorbitol Perovskia abrotanoides Adventitious roots Cryptotanshinone and [78]

Silver (Ag) Perovskia abrotanoides Adventitious roots Cryptotanshinone and [78]

Vitis vinifera Cell suspension Resveratrol [67]

Salvia castanea Hairy root Tanshinone [173]

Datura metel Hairy root Atropine [150]