**5.3 Elicitation as an effective strategy to enhance the productivity of** *in vitro* **cultures**

*In vitro* or *in vivo* cultured plants show physiological and morphological responses to physical, chemical, or microbial agents which are called elicitors. Therefore,

elicitation describes any processes that induce or enhance the synthesis of secondary metabolites to ensure plant survival and competitiveness [96, 97]. During *in vivo* growth, plant secondary metabolites are elicited in plant cells in response to environmental stresses as a defensive strategy against the abiotic agent or invading pathogen [2]. Elicitation effectiveness depends on several parameters, some of them are related to elicitor agents themselves, and others are related to the elicited *in vitro* cultured plant materials. The elicitor-related effects include elicitor type, concentration, and exposure duration. Cultures' age, cultivated line, medium composition, type, and concentration of growth regulators are essential parameters during the application of elicitation strategies. Hence, the application of factors, such as biotic or abiotic agents, that trigger the defense response in *in vitro*-cultivated plant materials enhanced the productivity of bioactive compounds [98].

Most of the used biotic elicitors are either exogenous or endogenous microbial agents but abiotic is a wide range of materials, mainly heavy metals [14, 99]. Methyl jasmonate, salicylic acid, yeast extract, chitosan, inorganic salts, UV radiation, or others can be used as elicitors to improve secondary metabolites production of the cultured plant materials [97, 100]. Citric acid, L-ascorbic acid, and casein hydrolysate were also used as elicitors to enhance the total phenolic content in the callus of *Rosa damascene* [49].

In the suspension culture of *Mentha pulegium*, when media were supplemented with yeast extract and salicylic acid, a significant increase of limonene, menthone, menthol, and α-pinene was detected [101]. Fifty different substances were detected in an *in vitro* cultured *Anemia tomentosa* upon jasmonic acid application, whereas 20 substances were only detected in wild-type plants [102]. Secondary metabolites production in callus, cell suspension, or hairy roots of *Ammi majus* L. were elicited by autoclaved lysate of cell suspension of *Enterobacter sakazaki* bacteria [103]. Anthraquinone production in *Rubia akane* cell culture was elicited by chitosan [104]. Genetically stable *in vitro* regenerated plants of *Capparis spinosa* were confirmed by RAPD analysis with a two-fold increase in flavonoid content than those of the wild plants when plants were regenerated under the influence of methyl jasmonate elicitor [105]. Elicitation of *Ambrosia artemisiifolia* hairy root cultures to produce thiorubrine A was dependent on cultures' age as well as elicitor concentration and exposure time. Maximum of eight-fold thiorubrine A production was achieved when 16-day-old cultures were elicited with 50 mg l-1 vanadyl sulfate elicitor for 72 h [106].

Abiotic stresses for a given period can be used as an elicitor. Temperature, light parameters (intensity, photoperiod, and wavelength), and water potential of the medium influence the fresh and dry biomass [15] as well as the concentration of active metabolites [107]. Any factor that affects the water stress of the media should affect growth and bioactive compound synthesis. The profound change in the culture water potential due to the addition of NaCl, mannitol, or polyethylene glycol can elicit the production of secondary metabolites [107]. The relationship between abiotic-nutritional deficiency stress and enhancement of the production of secondary metabolites was reported [108]. Deficiencies of nitrogen, phosphate, potassium, sulfur, or magnesium increase the production of phenolic compound accumulation in different plant species [109], which may be due to oxidative stress and modulation of the expression of some genes [110]. The combination between target gene overexpression and elicitors increased the yield of secondary metabolites. Across studied plant species, elicitors promoted the yield of secondary metabolites from 1.0 to a maximum of 2230-fold [100]. Abiotic elicitors were applied to enhance growth and ginseng saponin biosynthesis in *P. ginseng* hairy roots [111].

*DOI: http://dx.doi.org/10.5772/intechopen.105193 Application of Tissue Culture Techniques to Improve the Productivity of Medicinal Secondary...*

Specific microorganisms can be used for elicitor purposes [112]. It takes place through the co-cultivation of plant cells with microorganisms. Compared to non-elicited control tissues, coculture of *Aspergillus flavus* with *C. roseus* resulted in increases in vinblastine (7.88%) and vincristine (15.5%) concentrations [112]. Cocultivation between microorganisms and cultured plant tissue should avoid conditions that stimulate microorganism toxic components [12].

#### **5.4 Precursor feeding**

Under perfect and controlled conditions, *in vitro* cultured plants not only have a higher metabolic rate than differentiated or soil-grown plants but also compressed biosynthesis cycles in shorter periods of time. In addition, the addition of precursors and elicitors plays an important role in promoting the secondary metabolism of cells and tissues grown under well-controlled industrial conditions (PTC). Precursor feeding is a strategy that is based on the assumption that if intermediates of bioactive molecules are added at the beginning or during, the *in vitro* culture period, they can serve as a substrate to improve the production of secondary metabolites in cultured plant materials. Precursors refer to any compounds that can be converted by the *in vitro* cultured plant materials into secondary metabolites through biosynthetic pathways [14, 113], and they depend on the type and concentration of precursor, and addition timing [114]. According to the World Health Organization definition any plant that contains a substance that can be used for medicinal use or as a precursor to synthesize new or semi-synthetic pharmaceuticals as a medicinal plant. The addition of alanine precursor was used to stimulate the biosynthesis of plumbagin in *Plumbago indick* when it was added to the root cultures on the 14th day of cultivation along with sequential addition of Diaion HP-20 36 h after it was fed, this increased the target output 14 times [115]. Phenylalanine precursor was needed for the biosynthesis of silymarin in hairy roots of *Silymarin marianum* [116] or the biosynthesis of podophyllotoxin in the cell suspension cultures of *Podophyllum hexandrium* [117]. Combining elicitation with chitosan and precursor feeding with squalene was used to produce 27.49 mg/g DW withanolides [118].

Feeding the culture medium with organic compounds, such as vitamins or amino acids enhanced *in vitro* production of many secondary compounds. In callus and cell suspension cultures of *Centella asiatica,* amino acid feeding enhanced the production of triterpenes and asiaticoside [96]. Also, valine, threonine, and isoleucine enhanced adhyperforin production in shoot cultures of *Hyraceum perforatum* [119]. Feeding the suspension cultures of *C. roseus* with L- tryptophane or L-glutamine resulted in the production of the highest value of cell mass and indole alkaloids production [120]. Feeding the culture medium of *Spilanthes acmella* with casein hydrolysate and L-phenylalanine promoted biomass and scopoletin production [121]. Feeding squalene into culture medium of *C. asiatica* calli promoted production of madecassoside and asiaticoside [96]. In *Solanum lyratum* cell cultures, feeding with sterols such as cholesterol, stigmasterol or mixed sterols promoted the biosynthesis of solasodine, solasonidine, and solanine without effect on culture biomass [122].

The yield of salidroside was improved by feeding Rhodiola genus plants with an appropriate concentration of precursors and elicitors such as precursors, phenylalanine, tyrosol, and tyrosine [123]. Tyrosol feeding (0.5 mM) expressed the most obvious effect on salidroside content in the cell suspension cultures of *R. sachalinensis* [124]. When feeding the culture medium with precursors promoted the production of secondary metabolites without biomass accumulation, it needs a combination between precursors and elicitors to overcome the obstacle. This strategy was used to enhance the biosynthesis of sennoside A and B in callus cultures of *Cassia augustifolia* [125].

#### **5.5 High-yielding cell lines selection**

Genetic diversity within medicinal plants has great importance and can be used for plant improvement and the selection of an elite line. The selection of high biomass and metabolite(s) producing cell lines plays an important role in optimizing the productivity of *in vitro* cultivated plant materials. The yield of biomass and active metabolites may vary within varieties, genotypes, or populations of plant species [See 14]. The genotype has direct effects on the ability of the plant to produce valuable biomass and pharmaceutical compounds. To avoid high coast, the genotype with high yield and secondary metabolites contents should be carefully selected. For example, wright selection of *Pilocarpus microphyllus* resulted in the production of pilocarpine content ranging from 16.3 to 235.9 μg g-1 in dry weight [126], it was 15 times higher than the content found in wild plants.

To get a high yield of metabolites, Briskin [127] described the biotechnological methods for the selection of high-yielding cell lines in medicinal plants by addressing several topics, including media components, elicitation, immobilization, physical stress, and transformation. This means that the identification and establishment of high producing and fast-growing *in vitro* cultures are essential prerequisites, especially when the target secondary metabolite content of the selected cell line should be high. Selecting the higher-yielding cell lines was the essential step for optimizing the production of the anticancer drugs camptothecin [128].

Qualitative and quantitative estimation of active metabolites may show variability depending on the spatial and temporal changes that may happen during the process. Variation in secondary metabolites yield may be due to their repression or losses before or during the extraction processes. Consequently, the determined secondary metabolite value may not exactly indicate the actual content of secondary metabolite in a given tissue or plant species. Nevertheless, quantitative and qualitative methods can be applied to select high-yielding cell lines [14]. Selection of the high-yielding lines can be established by exposing the population of plant materials to toxic inhibitors, biosynthetic precursors, or stressful environments and followed by selecting cells that show higher production of targeted components [2]. Selection can be carried out using callus, cell suspension, or through any other *in vitro* culture procedure. In this regard, the answers to the following questions must be quite clear: Does diversity occur naturally or by using chemical, physical or biological substances that help in mutation to produce genetic diversity from which it can be selected? What are the methods used to identify and isolate the most qualitatively and quantitatively productive line?

#### **5.6 Overexpression of genes that control the production of bioactive compounds**

The production of secondary metabolites is a metabolic process that is influenced by several physicochemical factors. These factors can be controlled and optimized in large-scale production. Traditional mutagenesis programs have been used by the pharmaceutical industry for yield improvement of medicinal plants. Recently, the development of recombinant DNA technology has provided new and effective tools to obtain elite strains with high content of secondary metabolites through

#### *DOI: http://dx.doi.org/10.5772/intechopen.105193 Application of Tissue Culture Techniques to Improve the Productivity of Medicinal Secondary...*

overexpression of specific enzymes involved in their biosynthetic pathways aiming to increase the production levels and speed the metabolic processes [67, 96]. Consequently, plant genetics, recombinant DNA technologies, and PTC have developed to improve the ability of several medicinal plants to biosynthesize secondary metabolites efficiently.

To control the synthesis of certain natural products, the enzymes involved in the synthesis of these reactions and how they are influenced by *in vitro* culture conditions should be carefully determined. Niggeweg et al. [129] identified the enzymes that control the pathway of synthesis of an important bioactive compound through controlling these pathways. This control can be investigated on a gene expression and genome level [1] but it is not enough because it does not always give clear and specific information on the nature of the encoded enzyme that controls the intended reaction. Consequently, genomic studies have been used in combination with physiological and biochemical aspects to understand the biosynthetic pathways of specific secondary metabolites [1]. In this concern, metabolic engineering strategies concentrate on the stimulation of certain pathways over others by overexpressing certain genes.

Using PTC, key gene overexpression that involved in the biosynthetic of valuable biologically active compounds can be controlled leading to produce compounds in high quantity and quantity. For example, the overexpression of geranyl diphosphate synthase and geraniol synthase genes in *C. roseus* led to a significant improvement in plant production from monoterpene indole alkaloids of vinblastine and vincristine [130]. In periwinkle cell lines, overexpression of the strictosidine synthase (Str) gene resulted in tenfold activity than wild type leading to the accumulation of high content of ajmalicine, strictosidine, serpentine, tabersonine, and catharanthine [131]. Overexpressing tryptophan decarboxylase (Tdc) gene resulted in accumulation of TIAs (serpentine, catharanthine, strictosidine) more than wild type in transgenic cell suspension culture of periwinkle [132]. In addition, overexpression of H6H (hyoscyamine 6β-hydroxylase) from *Hyoscyamus niger* in *Atropa belladonna* hairy roots enhanced scopolamine production [133]. In addition, suppression of the rosmarinic acid synthase gene led to an increase in the plant content of 3,4-dihydroxyphenyllactic acid which led to improving the quality of rosmarinic acid in *Salvia miltiorrhiza* [134].

Bioactive secondary metabolites are under coordinated control of the biosynthetic genes, and transcription factors (TFs) play an important role in this regulation [135]. Transcriptional regulation means the change in gene expression levels by modulation of transcription rates. Studies on the regulation of the production of secondary metabolite pathways are focused on the regulation of structural genes through TFs [135]. For example, the expression of genes involved in TIAs (terpenoid indole alkaloids, such as vincristine and vinblastine) metabolic pathway is elicited by jasmonates, it is regulated biosynthesis of terpenoid indole alkaloid (TIAs) and artemisinin [135]. Jasmonate was demonstrated as a regulator of deacetylvindoline 4-O-acetyltransferase (DAT) expression [136]. Expressed DAT is involved in the biosynthesis of TIAs member-vindoline through transferring an acetyl group to deacetylvindoline for vindoline production. It was clear that most of the genes codded for TIA pathway enzymes are tightly regulated by specific TFs under the regulation of JAs but it is carried out in coordination with developmental growth stage and environmental factors [135].

TFs of TIA genes respond to JAs and/or other elicitors. In *C. roseus* a few TFs (CrORCA2, CrORCA3, CrBPF1, CrWRKY1, CrMYC1, and CrMYC2) have been characterized, two of them (ORCA2 and ORCA3) are positively influenced by JAs [137].

ORCA2 plays a critical role in the regulation of TIA metabolism where it regulates gene expression of both feeder pathways as well as STR and SGD, genes that codded for enzymes catalyzing the first two steps in biosynthesis of TIA [138]. In addition, ORCA3 overexpression resulted in the increase of some genes such as TDC, STR, and desacetoxyvindoline- 4-hydroxylase (D4H) leading to the accumulation of vinblastine and other metabolites in the TIA pathway [139]. Other TF such WRKY family that is induced by JAs is involved in TIA biosynthesis [140]. In *Catharanthus* hairy roots, overexpression of CrWRKY1 results in up-regulation of TIA pathway genes, especially the TDC gene. TF-CrWRKY1 binds the TDC promoter resulting in and transactivation of the TDC promoter in *Catharanthus* cells [141]. Preferential expression of CrWRKY1 and its interaction with other TFs (including CrORCAs and CrMYCs) play an essential role in the accumulation of vinblastine in *C. roseus* [135].

#### **5.7 Transformation**

The genetic transformation was used as a powerful tool to improve the productivity of secondary metabolites. In general, *Agrobacterium rhizogenes* was used to transfer genes in several dicotyledonous plants where roots are formed at the site of infection; what is called "hairy roots." Agrobacterium-mediated transformation technology may be better than direct gene transfer techniques including particle bombardment and electroporation [129]. Transformed hairy roots mimic the biochemical machinery of normal roots and are used to produce secondary metabolites where they are stable and have high productivity under growth regulators free culture [88]. Hairy roots transformed systems have great potential for commercial production of viable secondary metabolites and become a good alternative for raw plant materials.

Gene transfer using *Agrobacterium* can possibly be used to transfer DNA fragments that contain the genes of interest at higher efficiencies and lower cost. In *Raphanus sativus* L., a medicinal plant, plants formed hairy roots using *A. rhizogenes*, it was associated with the production of higher content of phenolic flavonoid and quercetin content compared to non-transformed plants [142]. Hairy roots were used for the production of phenolic acid, flavonoid, and wedelolactone from *Sphagneticola calendulacea* [143], tropane alkaloids of hyoscyamine, anisodamine, and scopolamine from *Scopolia lurida* [144].

*Bacopa monnieri* was transformed using *A. tumefaciens* with tryptophan decarboxylase and strictosidine synthase genes, which were obtained from *C. roseus.* Transformed tissues showed an increase in the terpenoid indole alkaloid pathway which led to an increase of 25-fold in tryptophan content in comparison with nontransformants [145]. Sharma et al. [146] used *A. tumefaciens* to transfer tryptophan decarboxylase and strictosidine synthase genes to *C. roseus*, it increased the content of terpenoid indole alkaloid metabolite due to the transient overexpression of these genes. In addition, several medicinal plants were subjected to genetic transformation including *Iphigenia indica* [88], *Artemisia annua* [57], *Aconitum heterophyllum* [100], *P. somniferum* L. and *Eschscholzia californica* [147]. *Solanum aviculare* [148], *Pueraria phaseoloides* [149], *Crataeva nurvala* [150], *Gymnema sylvestre* [151] and *Holostemma ada-kodien* [152] and *Araujia sericirfera* and *Ceropegia spp* [153].

#### **5.8 Scale-up production**

The application of PTC in medicinal plants can be scaled up using "bioreactors," which allow atomization and production of a high yield of medicinal secondary

#### *DOI: http://dx.doi.org/10.5772/intechopen.105193 Application of Tissue Culture Techniques to Improve the Productivity of Medicinal Secondary...*

products [154]. Therefore, scale-up production is a bioreactor application for the cultivation of plant cells on large-scale aiming for the mass production of valuable bioactive compounds. Also, bioreactor-based micropropagation was found to increase shoot multiplication for the commercial propagation of *B. monnieri* plants and maximize the content of bacosides in shoot biomass using an airlift bioreactor system [154]. Production of secondary metabolites using *in vitro* culture techniques is recommended strategy, especially when studying morphological and physiological processes associated with metabolites biosynthesis is necessary [155].

Cell suspension offers the wright combination of physical and chemical environments that must be used in the large-scale production of secondary metabolites in the bioreactor process [156]. Consequently, scale-up production in the bioreactor was used to expand the production of secondary metabolites from research to the industrial level. Systems of various sizes and features of bioreactors were created and applied for the mass production of secondary metabolites [157]. The application of plant tissue culture techniques in bioreactors for scale-up production facilitates obtaining some expensive pharmaceuticals that are synthesized in low quantity during *in vitro* or *in vivo* cultures. Since scale-up production of skikonin substance was achieved using bioreactors by Tabata and Fujita [158], other successful scale-up productions were obtained such as ginseng [159] and taxol [160].

Bioreactor operating system should provide efficient oxygen and nutrient supply, homogenous distribution of cultivated plant materials, and other factors that ensure optimal biomass and metabolite production [161]. While most of these bioreactors rely on cell suspension cultures, few of which are rely on differentiated tissues such as somatic embryos and hairy roots [162]. Application of suspension culture facilitates metabolites isolation [157].

For scale-up production, automation becomes an essential prerequisite, where it controls the pH of the culture area, culture viscosity, osmolarity, temperature, redox potential, oxygen supply, production of carbon dioxide, nutrients, weight, and liquid levels, and follows the rate of cell density. This automation needs sensors and monitoring systems that ensure mass production of pharmaceuticals and monitoring of physical, chemical, and biological parameters [163].

Perfusion cultivation is a system where continuous feeding of fresh media into a bioreactor system and removal of cells-free media were carried out in a modified bioreactor. The aim of this type of bioreactor and perfusion cultivation is to scalingup the production of pharmaceutical compounds using plant cell, tissue, and organ cultures. The perfusion system offers a great advantage where it overcomes nutrient depletion and accumulation of growth inhibitors within the cultivated system, and it resulted in the promotion of biomass and pharmaceutical compounds. Semicontinuous perfusion was established in *Anchusa officinalis* where it was carried out in the shake flasks with a manual exchange of media. It resulted in the promotion of more than two-fold cell density and rosmarinic acid production in comparison to batch cultures [164].

Advances in immobilization and scale-up production techniques increase the applications of plant cell cultures for the purpose of producing high added value secondary compounds such as compounds with chemotherapeutic or antioxidant properties. For example, cell cultures of *Plumbago rosea* were immobilized using an MS medium containing 10 mM CaCl2 and calcium alginate for the production of important medicinal compounds, such as plumbagin [165]. Their studies indicated the impact of immobilization on the increased accumulation of plumbagin where

immobilization in calcium alginate resulted in enhancement of plumbagin production up to three folds compared with that of control [156].
