VEER CHANDRA SINGH GARHWALI UTTARAKHAND

UNIVERSITY OF HORTICULTURE AND FORESTRY

College of horticulture, Bharsar

Session-2015-16

Course: map 509:- master’s seminar

Topic:- role of elicitors in production of plant

secondary metabolites under in-vitro

condition

Submitted To: submitted by:

Dr. ajaya paliwal yashwant singh tariyal

(seminar incharge) id. No.:- uuhf/13162

Dept. of crop improvement Department of plantation crops, spices, medicinal and aromatic plants

ABSTRACT

Bioactive compounds extracted from plants are widely used. Currently, most of these secondary metabolites are isolated from wild or cultivated plants because their chemical synthesis is either extremely difficult or economically infeasible. Biotechnological production in plant cell cultures is an attractive alternative, but to date this has had only limited commercial success because of a lack of understanding of how these metabolites are synthesized. The principle advantage of this technology is that it may provide continuous, reliable source of plant pharmaceuticals and could be used for the large-scale culture of plant cells from which these metabolites can be extracted (Mulabagal and Tsay, 2004). Biotechnological applications of plant cell cultures presents the most updated reviews on current techniques in plant culture in the field. The evolving commercial importance of the secondary metabolites has in recent years resulted in a great interest, in secondary metabolism, and particularly in the possibility to alter the production of bioactive plant metabolites by means of cell culture technology. Studies on the production of plant metabolites by callus and cell suspension cultures have been carried out on an increasing scale since the end of the 1950's. The prospect of using such culturing techniques is for obtaining secondary metabolites, such as active compounds for pharmaceuticals and cosmetics, hormones, enzymes, proteins, antigens, food additives and natural pesticides from the harvest of the cultured cells or tissues (Terrier et al., 2007). Plants and/or plant cells in vitro, show physiological and morphological responses to microbial, physical or chemical factors which are known as ‘elicitors’. Elicitation is a process of induced or enhanced synthesis of secondary metabolites by the plants to ensure their survival, persistence and competitiveness (Namdeo, 2007). Elicitation is the induction of secondary metabolite production by either biotic or abiotic treatments. Nowadays, the use of pathogenic and non-pathogenic fungal preparations and chemicals as elicitors has become one of the most important and successful strategies to improve secondary metabolite production in plant cell culture (Baldi et al., 2009).

Yashwant Singh Tariyal (Yashutariyal50@gmail.com)

(Author)

Introduction

Medicinal plants are the most exclusive source of life saving drugs for the majority of the world’s population (Balandrin and Klocke, 1988). These compounds belong to a group collectively known as secondary metabolites. Studies on plant secondary metabolites have been increasing over the last 50 years. The molecules are known to play a major role in the adaptation of plants to their environment (Ramachandra Rao and Ravishankar, 2002). In recent years, traditional system of medicine has become a topic of global importance. Although modern medicine may be available in developed countries, herbal medicines (Phyto-pharmaceuticals) have often maintained popularity for historical and cultural reasons.

Many of the plant species that provide medicinal herbs have been scientifically evaluated for their possible medical applications. It has been mentioned that natural habitats for medicinal plants are disappearing fast and together with environmental and geopolitical instabilities; it is increasingly difficult to acquire plant-derived compounds. This has prompted industries, as well as scientists to consider the possibilities of investigation into cell cultures as an alternative supply for the production of plant pharmaceuticals. Advances in biotechnology particularly methods for culturing plant cell cultures, should provide new means for the commercial processing of even rare plants and the chemicals they provide. These new technologies will extend and enhance the usefulness of plants as renewable resources of valuable chemicals. There has been considerable interest in plant cell cultures as a potential alternative to traditional agriculture for the industrial production of secondary metabolites (Dicosmo and Misawa, 1995). Plant cell culture technologies were introduced at the end of 1960s as a possible tool for both studying and

producing plant secondary metabolites. Different strategies using cell cultures systems have been extensively studied with the objective of improving the production of bioactive secondary metabolites. Cell culture systems could be used for the large scale culturing of plant cells from which secondary metabolites can be extracted. The advantage of this method is that it can ultimately provide a continuous, reliable source of natural products (Mulabagal and Tsay, 2004).

The objectives of many industries are to develop plant cell culture techniques to the stage where they yield secondary products more cheaply than extracting either the whole plant grown under natural conditions or synthesizing the product. Although the production of pharmaceuticals using plant cell cultures have been highlighted, other uses have also been suggested as new route for synthesis, for products from plants difficult to grow, or in short supply, as a source of novel chemicals and as biotransformation systems. It is expected that the use, production of market price and structure would bring some of the other compounds to a commercial scale more rapidly and in vitro culture products may see further commercialization. Recent research results indicate that plant cell suspension cells can be used for recombinant protein production under controlled conditions (Fischer et al., 1999).

Plant secondary metabolites

Plant secondary metabolites can be defined as compounds that have no recognized role in the maintenance of fundamental life processes in the plants that synthesize them, but they do have an important role in the interaction of the plant with its environment. (Namdeo, 2007). Secondary metabolites are substances which are produced by plants as defense chemicals. Their absence does not cause bad effects to

the plants. They include alkaloids, phenol, steroids, essential oils, lignin, resins and tannins etc. These compounds are biosynthetically derived from primary metabolites. Secondary metabolites or secondary compounds are compounds that are not required for normal growth and development but, they are required for survival of plant under natural condition, and are not made through metabolic pathways common to all plants. In plant kingdom they are limited to occurrence and may be restricted to a particular taxonomic group genus, species or family. The production of these compounds is often low (less than 1% dry weight) and depends greatly on the physiological and developmental stage of the plant thus, making extraction and purification difficult (Kumar et al., 2014).

Importance of plant secondary metabolites

They are the major component of plant defense mechanism against biotic as well as abiotic stresses such as; herbivorous animals, insects, pest and pathogen as well as humans (biotic) and environmental constraints such as; light, temperature, humidity, alkalinity, salinity and acidic condition of soil etc. (abiotic). For human use they are the major source of various chemical components for industrial use these secondary metabolites are used in preparation of various dyes, medicine, and therapeutic use. Used for making agrochemicals such as insecticides pesticides fungicides etc. these are the basic raw material for many industries such as perfume, cosmetics, food colour and flavoring agent etc.

Classification of plant secondary metabolites (Mahmoud and Croteau, 2002)

o Nitrogenous secondary metabolites.

Alkaloids

Non-protein amino acids (NPAAs)

Amines

Cyanogenic glycoside

Glucosinolates

Alkamides

Lectins, peptides, polypeptide

o Non-Nitrogenous secondary metabolites

Terpenes including iridoids

Steroids, saponins

Flavonoids, tannins

Phenylpropanoids, lignin, coumarins, lignans

Polyacetylenes, fatty acid, waxes

Anthraquinones and others polyketides

Carbohydrates, organic acids

In-vitro production of plant secondary metabolites

The process of in-vitro culture of cell for the large scale production of secondary metabolites involves the following aspects:

1. Selection of cell line for high yield of secondary metabolites.

2. Large scale cultivation of plant cells. 3. Media composition and effect of nutrients. 4. Elicitors induced production of secondary metabolites. 5. Effect of environmental factor. 6. Biotransformation using plant cell culture. 7. Secondary metabolite release and analysis.

Culture system used for secondary metabolite production

The following four different culture systems are widely used:

1. Free cell suspension culture: mass cultivation of plant cell is most frequently carried out by cell suspension culture. Care should be taken to achieve good growth of cell and efficient formation of the desired secondary metabolites. Many specially designed bioreactors are used for free cell suspension cultures. Some of these are:

a. Batch bioreactor b. Continuous bioreactors c. Multistage bioreactor d. Airlift bioreactor e. Stirred tank bioreactor

Two aspects have to be considered for good success of suspension cultures

a. Adequate and continuous oxygen supply

b. Minimal generation of hydrodynamic stresses due to aeration agitation.

2. Immobilization cell cultures: plant cells can be made

immobile or immovable and used in culture system. The cells are physically immobilized by entrapment. Besides individual cells, it is also possible to immobilize aggregate cell or even calluses. Homogenous cell suspension culture is most suitable for immobilization.

3. Two phase system culture: Plant cells can be cultivated in an aqueous two phase system for the production of secondary metabolites. In this technique, the cells are kept apart from the product by separation in the bioreactor. This is advantageous since the product can be removed continuously.

4. Hairy root culture: These cultures are used for the production of root associated metabolites. In general, these cultures have high growth rates and genetic stability. For the production of hairy root cultures, the ex-plant material is inoculated with cells of the pathogenic bacterium, Agrobacterium rhizogen. the organism contain root inducing plasmid that cause genetic transformation of plant tissues, which finally result in hairy root culture. Hairy root produced by plant tissues have metabolite features similar to that of normal roots.

Hairy root cultures are most recent organ root culture system and are successfully used for the commercial production of secondary metabolites

Some of the major advantages of in-vitro production of plant secondary metabolites are as follows:

o Helpful in conservation of threatened plant species.

o Compounds can be produced under controlled conditions as per market demand.

o Culture systems are independent of environmental factors, seasonal variations, pest and microbial diseases and geographical constraints.

o Cell growth can be controlled to facilitate improved product formation and consistent quality of the product.

o It is very useful in the plants which are difficult and expensive to grow in field.

o The production time is less and labour cost is minimal.

o In vitro production of natural material is cheaper is compared to synthetic production.

o Recovery of the product will be easy.

o Mutant cell lines can be developed for the production of novel compounds of commercial importance which are not normally found in plants.

o Biotransformation reaction (converting specific substrate into valuable product) can be carried out with certain cultured cells.

Considering the advantage of in-vitro production, about 20-30% of medicine for human use and various chemical materials for industrial purpose are obtained from plant tissue culture. In general it is cheaper compared to synthetic

production. However, there are certain limitations associated with tissue culture.

o In general, in-vitro production of secondary metabolites is lower when compared to the intact plant.

o Many time secondary metabolites are produced in differentiated tissue/organ. In such cases the culture cells which are non-differentiated produce less quantity.

o Cultured cells are genetically unstable and may undergo mutation this may cause in reduced production, as culture ages.

o Vigorous stirring is necessary to prevent aggregation of cell this may often damage cells.

o Strict aseptic conditions have to be maintained during culture technique.

Elicitors and Elicitation

An ‘elicitor’ may be defined as a substance which, when introduced in small concentrations to a living cell system, initiates or improves the biosynthesis of specific compounds. Elicitation is the induced or enhanced biosynthesis of metabolites due to addition of trace amounts of elicitors (Radman et al., 2003).

These are compounds which stimulating any type of physiological abnormality of plant. This broader definition of elicitors includes both substances of pathogen origin (exogenous elicitors) and compounds released from plants by the action of the pathogen (endogenous elicitors). Elicitors could be used as enhance of plant secondary-metabolite synthesis and could play an important role in biosynthetic

pathways to enhanced production of commercially important compounds.

The secondary metabolites are released due to defense responses which are triggered and activated by elicitors, the signal compound of plant defense responses.

Classification of Elicitors

Elicitors are classified as physical or chemical. On the basis of nature elicitors can be divided into two types Biotic and Abiotic. The biotic elicitors have biological origin, derived from the pathogen or from the plant itself while abiotic elicitors have not a biological origin and are grouped in physical factor and chemical compounds. The first biotic elicitor was discovered in 1968. Further on the basis of plant elicitor interaction it may be classified into race specific and general elicitors. Elicitation of plant cell culture system may be promising as it showed favorable results in fermentation of antibiotics and many other fermented products. Though, elicitation enhances secondary metabolism in plants or plant cells in vitro. This provides an opportunity for intensive research in the field of biosciences for exploitation of plant cells for the production of secondary metabolites.

Elicitors

Physical Chemical

Light, Temperature Abiotic Biotic

ComplexDefined Composition

Metal Ions, Oxalates

Yeast Cells Wall, Mycelia Cell Wall, Fungal Spores Carbohydrates, Proteins,

lipids, Glycoprotein, Volatiles

Radman et al. (2003)

Classification of elicitors

Effect of physical factors

Physical factors namely light, incubation temperature, pH of the medium and aeration of culture influence the production of secondary metabolites in cultures.

Effect of light:

Light is absolutely essential for the carbon fixation (photosynthesis) of field grown plants. Since the carbon fixation is almost absent or very low in plant tissue cultures, light has no effect on the primary metabolism.

However, the light mediated enzymatic reactions indirectly influence the secondary metabolite formation. The quantity of light is important. Some of the examples are:

1. Blue light enhances the anthocyanin production in Haplopappus gracilis cell suspension culture.

2. White light increases the formation of anthocyanin in culture of Catharanthus roseus Daucus carota and Helianthus tuberosus.

3. White and blue light inhibits the naphthoquione biosynthesis in callus culture of Lithospermum erythrorhizon.

Effect of incubation temperature:

The growth of cultured cells is increased with increase in temperature upto an optimal temperature (25-30oC). However, at least for the production some secondary metabolites lower temperature is advantageous. For instance in Catharanthus roseus cultures, indole alkaloid production are increased by two fold when incubated at 16oC instead of 27oC.

Effect of pH of the medium:

For good growth of culture, the pH of the medium is in the range of 5 to 6. There are reports indicating that pH of the medium influence the formation of secondary metabolites. Such as production of anthocyanin by culture of Daucus carota was much less whenincubated at pH 5.5 than at pH 4.5. This indicates degradation of anthocyanin at higher pH.

Aeration of culture:

Continuous aeration is needed for good growth of cultures and also for efficient production of secondary metabolites.

Effect of chemical factors

Nitrogen

The standard culture medium usually contains a mixture of nitrate and ammonia as nitrogen source. Majority of plant cell can tolerate high level of ammonia. The cultured cells utilize the nitrogen for the biosynthesis of amino acids protein and nucleic acids. The nitrogen containing primary metabolites directly influence the secondary metabolites.

In general, high ammonium ion concentration inhibits the secondary metabolite formation while lowering of ammonium ion concentration increases. It is reported that addition of KNO

3 and NH

4NO

3 inhibit anthocyanine by 90% and alkaloid

by 80% production.

Phosphate:

Inorganic phosphate is essential for photosynthesis and respiration. In addition, many secondary metabolites are produced through phosphorylated intermediates, which subsequently phosphate e.g., phnylpropanoids, terpenes, terpenoids. In general, high phosphate level promotes cell growth and primary metabolism while low phosphate concentrations are beneficial for secondary metabolite formation.

1. Increase phosphate concentration increase alkaloids production such as; anthraquinone in Catharanthus roseus and Morinda citrifolia and diosagenin in Dioscorea sp.

2. Decrease phosphate level in the medium increases the formation of anthocyanine and phenols in Catharanthus roseus, alkaloids in Peganum harmala and solasodine in Solanum lanciatum.

3. Phosphate concentration has no effect on alkaloid production of berberis Sp.

Plant hormones

There are various plant hormones which act as a elicitors. The common plant hormones like Salicyclic acid (SA) and Jasmonic acid (JA) are key signals for defense gene expression , In which SA regulates resistance to pathogens like bacterial, fungal and viral, Whereas JA regulates the production of

proteins by the octadecanoid pathway. SA and JA are both synthetic mimics that can be applied externally to induce same metabolic changes that regulate resistance against pathogen. The biochemical pathways of both SA and JA are useful in the plant elicitation process (Angelova et al., 2006).

Carbohydrates

In plant tissue culture, there are different carbohydrates useful in the overproduction of secondary metabolites. Albersheim (1977) first isolated to oligosaccharides that regulate variety of plant defense gene. In tobacco cell cultures the carbohydrates elicitors are induce the signal transfer with regard to calcium influx and production of H2O2 (Negeral and Javelle, 1995).

Eliciators in plant tissue culture technology are used for various purposes such as follows:

Elicitors

Plant Tissue Culture

Investigation of Plant Defense Mechanisms

Investigation of Enzymology of

Secondary Metabolism

Investigation of Regulation of

Secondary Metabolism

Increase Yield of Target Substance

Utilization of elicitation of plant tissue cultures in various areas of research.

Veersham, 2004

Mechanism of elicitation in plant cells

‘Elicitor’ for a plant refers to chemical from various sources that can trigger physiological and morphological responses and phyto-chemical accumulation. It may include abiotic elicitors such as metal ions and inorganic compounds and biotic elicitors from fungi, bacteria or herbivores, plant cell wall fragments as well as chemicals that are released at attack site by plants upon pathogen or herbivore attack. It is well known that the treatment of plants with elicitors or attack by incompatible pathogen causes an array of defense reactions, including the accumulation of a range of plant defensive secondary metabolites in intact plants or in plant cell cultures. Even after the intensive research on the effect of biotic and abiotic elicitors on the production of secondary metabolites in plants, the exact mechanism of elicitation is poorly understood. Various mechanisms in this regard have been hypothesized like messenger Ca2+, factors affecting cell membrane integrity, inhibition/ activation of intracellular pathways and changes in osmotic stress etc. Some groups highlighted the rapid changes in protein phosphorylation patterns and protein kinase activation as mechanism of elicitation. While others observed mitogen-activated protein kinase (MAPK) stimulation and G-protein activation. Cytoplasm acidification caused by H+-ATPase inactivation, whereas the decrease in membrane polarization, extracellular increase of pH has been reported in elicitor treated plant tissues. The production of ROS such as the superoxide anion and H

2O

2 that might have a direct antimicrobial effect as well

as contributing to the generation of bioactive fatty acid derivatives. Similar observation of ROS involvement in the cross-linking of cell-wall-bound proline-rich proteins H

2O

2 can

act as a secondary messenger and it is involved in the

transcriptional activation of defense genes. In another hypothesis, accumulation of defense-related proteins pathogenesis related proteins such as chitinases and glucanases, endo-polygalacturonases that contribute to the release of signaling pectic oligomers (endogenous elicitors), hydroxyproline-rich glycoproteins, and protease inhibitors. Hypersensitive response to cell death at the infection site was observed by some groups. Transcriptional activation of the corresponding defense response genes for elicitation process has been reported. The exact mechanism of elicitation is the study of these events and their interconnection and inter-correlation between them is highly complex and is still under investigation. All elicitors do not follow the same sequence of events but varies with their origin, specificity, concentration, physiochemical environment, stage of their growth cycle, nutritional uptake etc. Characteristics of Elicitors the enhanced production of the secondary metabolites from plant cell cultures through elicitation has opened up a new area of research which could have important economical 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, quality of cell wall materials, substantial enhancement of product accumulation etc (Namdeo, 2007).

STUDIES RELATED TO THE TOPIC:

Study 1:

Meena and her co-workers (2014) studies the effect of β–phenylalanine on growth of tissue and production of quercetin on liquid culture of Citrullus colocynthis.their result indicate a gradual increase in growth index upto six weeks; after that it declined. GI increased when suspension culture was fed with

phenylalanine (25-50 mg/100 ml). Further increase in phenylalanine concentration (75-100 mg/100 ml) decreased GI. Maximum GI (4.85 mg/g dw) as observed in 6 weeks old tissue fed with 50 mg/100 ml of phenylalanine and minimum GI (0.95 mg/g dw) was observed in 2 weeks old tissue fed with 100 mg/100 ml of phenylalanine.

Figure 1:- Effect of phenylalanine on growth indices and production of quercetin in 6 weeks old tissue.

Total quercetin content (free and bound quercetin) showed a marked increase in tissue fed with phenylalanine upto 50 mg PA/100 ml of medium thereafter it declined gradually and was rather low in tissues grown on the medium fed with 100 mg PA/100 ml of medium but this amount was still higher as compared to the amount obtained from tissues grown on control medium.

Maximum quercetin content was found in six weeks old callus cultures. Maximum (7.25 mg/g dw) quercetin was observed in callus tissue fed with 50 mg PA/100 ml medium and minimum (4.08 mg/g dw) in tissues grown on media fed with 100 mg PA/100 ml of medium (as compared to control 3.05 mg/g dw).

Table 1:- Effect of β-phenylalanine on growth of tissue and production of quercetin on liquid culture of Citrullus Colocynthis.

Study II

Usha and her co worker (2015) studied the production of secondary metabolites from callus cultures of Centella asiatica (L.) Urban and reported that methyl jasmonate and salisylic acid can be used as elictors to enhance the secondary metabolites in C. asiatica and the optimum concentrations of these elicitors is 100 μM and concentration beyond this is inhibitory for the production of secondary metabolites in this species.

Table 2:- Effect of elicitors on the secondary metabolites content in leaf derived callus cultures of C. asiatica.

Case 3:

Barmukh and his co-worker (2009) studies the effect of three biotic elicitor independently and reported that incorporation of increasing concentration of homogenate of biotic elicitors, Aspergillus niger Alternaria sps. Fusarium monoliforme, in the medium result in higher hyoscyamine and scopolamine accumulation with the reduced growth of culture. The growth index decline gradually with the increase concentration of the fungal homogenate in medium. However, among the same concentration of elicitors homogenates, no significant difference was observed for the growth index. Culture treated with 1.0gL-1 of Aspergillus niger homogenate results in higher hyoscyamine (1.77 mg/g dw) and scopolamine (0.087 mg/g dw) production than that of Alternaria sps. and Fusarium monoliforme. The result indicates that the

Aspergillus niger homogenate is favorable for promoting the accumulation of tropane alkaloids in Datura metel L.

Elicitor (g L-1)

Growth Index

Hyoscyamine(mg/g dw)

Scopolamine(mg/g dw)

Aspergillus niger

0.0 5.79 1.39±0.004 0.069±0.001

0.1 6.32 1.46±0.003 0.077±0.0004

0.5 5.61 1.75±0.005 0.082±0.0005

1.0 5.07 1.77±0.005 0.087±0.001

1.5 4.34 1.63±0.004 0.081±0.001

Table: 3 (a)-:Effect of biotic elicitor on the alkaloid content in the callus and root culture of Datura metel L.

Barmukh et al. 2009

The values represent the mean ± SE of three independent experiment each performed on 21 replicates.

Elicitor (g L-1)

Growth Index

Hyoscyamine(mg/g dw)

Scopolamine(mg/g dw)

Alternaria sps.

0.0 5.79 1.39±0.004 0.069±0.001

0.1 5.96 1.43±0.006 0.074±0.001

0.5 5.43 1.59±0.004 0.079±0.0008

1.0 4.89 1.64±0.003 0.084±0.001

1.5 4.24 1.52±0.004 0.078±0.001

Table: 3 (b)-:Effect of biotic elicitor on the alkaloid content in the callus and root culture of Datura metel L.

The values represent the mean ± SE of three independent experiment each performed on 21 replicates.

Barmukh et al. 2009

Elicitor (g L-1)

Growth Index

Hyoscyamine(mg/g dw)

Scopolamine(mg/g dw)

Fusarium monoliforme

0.0 5.79 1.39±0.004 0.069±0.001

0.1 6.14 1.44±0.003 0.072±0.014

0.5 5.61 1.63±0.005 0.075±0.0007

1.0 5.25 1.66±0.004 0.081±0.0006

1.5 4.92 1.55±0.006 0.072±0.0006

Table: 3 (c)-:Effect of biotic elicitor on the alkaloid content in the callus and root culture of Datura metel L.

The values represent the mean ± SE of three independent experiment each performed on 21 replicates.

Barmukh et al. 2009

Study 4:

Mathew and Sankar 2014 studies the comparison of major secondary metabolites quantified in elicited cell cultures, non-elicited cell cultures, callus cultures and field grown plants. The two elicitor used are methyl jasmonate and chitosan and their combination (Elicitation 1 - MeJA 25μM, 12 h, Elicitation 2 - Chitosan 200 mg/L, 12 h, Elicitation 3 - MeJA 25μM + chitosan 200 mg/L, 8 h).

Ocimum basilicum L.

When quantified for total alkaloid content the highest (0.22 ± 0.01 gm/gm DW) was observed again in the cell culture elicited with both MeJA and chitosan (MeJA 25μM + chitosan 200 mg/L, 8 h). The second highest total alkaloid content was observed in the MeJA elicited cell culture (19 ± 0.01 gm/gm DW). Alkaloid content was observed to be almost the same for callus culture, non-elicited cell culture, chitosan elicited cell culture and the plant material of O. basilicum.

In the case of total terpenoid content (Figure 1c), cell culture elicited with both MeJA and chitosan (MeJA 25μM + chitosan 200 mg/L, 8 h) was again observed to have the highest which was found to be 0.45 ± 0.02 gm/gm DW. Low alkaloid content was observed for callus culture (19 ± 0.01 gm/gm DW), chitosan elicited cell culture (19 ± 0.01 gm/gm DW) and the leaves (19 ± 0.02 gm/gm DW) of O. basilicum.

Ocimum sanctum L.

Cell culture elicited with MeJA was again found to show the highest (0.33 ± 0.01 gm/gm DW) when quantified for total alkaloid content. The lowest was observed in the cell culture elicited with chitosan (14 ± 0.01 gm/gm DW).

In the case of total terpenoid content, cell culture elicited with MeJA was again found to show the highest (0.43 ± 0.03 gm/gm DW). The lowest was observed for the cell culture elicited with both the elicitors (0.12 ± 0.03 gm/gm DW).

Ocimum gratissimum L.

For total alkaloid content (Figure 1b), cell culture elicited with chitosan was again found to show the highest (0.45 ± 0.01 gm/gm DW). The lowest was observed in the callus culture, which was found to be 0.15 ± 0.01 gm/gm DW.

For total terpenoid content (Figure 1c), the highest was again observed in the chitosan elicited cell culture, which was found to be 0.42 ± 0.04 gm/gm DW. The lowest (14 ± 0.02 gm/gm DW) was again observed in the callus culture.

O. basilicum L.: Elicitation 1 - MeJA 25μM, 12 h, Elicitation 2 - Chitosan 200 mg/L, 12 h, Elicitation 3 -MeJA 25μM + chitosan 200 mg/L, 8 h

O. sanctum L.: Elicitation 1 - MeJA 25μM, 48 h, Elicitation 2 - Chitosan 50 mg/L, 24 h, Elicitation 3 -MeJA 100 μM + chitosan 200 mg/L, 4 h

O. gratissimum L.: Elicitation 1 - MeJA 50 μM, 8 h, Elicitation 2 - Chitosan 50 mg/L, 24 h, Elicitation 3 - MeJA 25 μM + chitosan 100 mg/L, 24 h

Figure: 1:-Quantification of total alkaloid content in Ocimumspecies

Mathew and Sankar 2014

Figure: 2:-Quantification of total terpenoid content in Ocimum species.

O. basilicum L.: Elicitation 1 - MeJA 25μM, 12 h, Elicitation 2 - Chitosan 200 mg/L, 12 h, Elicitation 3 -MeJA 25μM + chitosan 200 mg/L, 8 h .

O. sanctum L.: Elicitation 1 - MeJA 25μM, 48 h, Elicitation 2 - Chitosan 50 mg/L, 24 h, Elicitation 3 -MeJA 100 μM + chitosan 200 mg/L, 4 h .

O. gratissimum L.: Elicitation 1 - MeJA 50 μM, 8 h, Elicitation 2 - Chitosan 50 mg/L, 24 h, Elicitation 3 - MeJA 25 μM + chitosan 100 mg/L, 24 h .

Mathew and Sankar 2014

Conclusion

According to WHO report about 70-80% 0f world population depends on the herbal based drugs. The demand for the plant and their purified product is continuously increasing day by day. This puts a lots pressure on the agricultural land for the production of medicinal plants among which some of the plants are difficult or expensive for cultivation due to various biotic abiotic and economical factors. However, the plant tissue culture techniques are the best alternatives for the production of economically important secondary metabolites and elicitors plays a key role in increasing the in-vitro production of these compounds with consistent quality as per market demand.

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