**4. Application of** *in vitro* **culture techniques for the production of pharmaceuticals**

The synthetic capacity of secondary metabolites of the dedifferentiated tissue often differs substantially from that of differentiated one, both quantitatively and qualitatively. The differences in synthetic capacities are a direct response to differences in enzyme patterns between differentiated and undifferentiated tissues, they are mirrors for gene expression of these tissues. The culture of differentiated plant materials often shows biochemical and genetic stability, it offers a high-productivity system that does not need wide-ranging optimization. For example, the major alkaloid (vindoline) is scarcely produced by *Catharanthus roseus* suspension cultures but shoot cultures produce it in high quantity [60]. In addition, while the callus culture of *Taraxacum officinale* synthesizes and accumulates α and γ-amyrins, differentiated tissue synthesizes and accumulates taraxasterol and lupeol [61]. The previous studies indicate that different classes of secondary metabolites need different phases of cell or tissue differentiation.

Generally, *in vitro* conditions can be easily modulated to enhance the synthesis of secondary metabolites through modulation of the pathway of primary metabolism in plants. The *in vitro* obtained compounds are important as dyes, drugs, cosmetics, flavors, food additives, perfumes, agrochemicals, etc. Some of these compounds, such as flavors, fragrances, and colorants, cannot be produced by microbial cells or chemically synthesized but they can be synthesized by plant cell culture systems [62]. Several reports indicated that *in vitro* cultures were found to be more efficient than whole plants for the formation of bioactive secondary metabolites such as ajmalicine, ajmaline, anthraquinones, benzylisoquinoline alkaloids, berberine, bisoclaurine, coniferin, diosgenin, ginseng, ginsenoside, glutathione, nicotine, rosmarinic acid, raucaffricine, shikonin, taxol, terpentine, tripdiolide, and ubiquinone-10 [1, 2, 14, 62].

Under aseptic conditions, cultured plant materials can be used to generate bioactive or secondary metabolites, including flavonoids, alkaloids and other phenolics, terpenoids, saponins, steroids, tannins, glycosides, colorants, fragrances, and volatile oils [14]. Production of high-value active secondary metabolites at industrial levels, such as shikonin, berberine, and sanguinarine, was fulfilled from cell cultures of *Lithospermum erythrorhizon*, *Coptis japonica,* and *Papaver somniferum*, respectively [63]. Secondary bioactive metabolites in *in vitro* cultured *Swertia chirayita* were higher than *in vivo* plants [2]. The more antimicrobial property of the *in vitro* regenerated plant products was related to more bioactive metabolites. In addition, Manivannan et al. [20] reported that since the contents of phytochemicals in seed and *in vitro* derived plants were similar, the *in vitro* plantlets can be used as alternate for the seed grown plants for the production of bioactive metabolites. Also, acacetin (an individual flavonoid) was slightly increased in *in vitro* grown plantlets than that of *in vivo* grown plants due to the artificial conditions of the *in vitro* culture and modulation of endogenous hormone [20].

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

Pharmaceutical compounds that are obtained from *in vitro* cultured plant materials may be more easily extracted and purified due to the absence of significant amounts of pigments, thus resulting in lower manufacturing expenses [64]. Control of the production of secondary metabolites can be carried out using *in vitro* culture techniques. For example, low biomass and hypericin production of *Hypericum perforatum* shoots was improved by prolonging the time of culture for more than 30 days [65].

### **5. Strategies are used to improve secondary metabolites production**

The biosynthesis of secondary metabolites using unorganized cultured cells or organized organs, such as roots, can be enhanced by altering the environmental conditions or selecting an elite variant clone [66]. There are many procedures that can be controlled to increase the productivity of *in vitro* cultured medicinal plants from the active substances with medicinal effects, and this is what will be discussed in this chapter.

#### **5.1 Culture media optimization**

To understand factors that control the biosynthesis of pharmaceutical compounds by cultured plant materials, studies on gene expression, enzyme activity, and signal transductions were carried out [12, 14]. The establishment of desired productivity of the PTC needs optimization of overall culture conditions to enhance both culture biomass and metabolites productivity. For example, while sulfate and ammonium nitrate ions increased the colchicine content of *Gloriosa superba* callus, a higher concentration of phosphate and calcium decreased alkaloid biosynthesis [67]. The differences in the composition of various PTC media formulations affect on water potential of the cultural environment [68]. Then, different media exerted different values of water potential. *In vitro* culture of certain medicinal plant materials on different media expresses different values of biomass and secondary metabolites. Medium selection is a major step in optimizing the culture conditions to produce an abundance of plant matter capable of producing an abundance of biological compounds [69]. Through media optimization of the *in vitro* cultured medicinal plants, the chemical composition is changed, the content of toxic compounds is reduced and novel chemical compounds may be formed [70]. In general, media optimization is an essential prerequisite to enhancing the production of antioxidants and other valuable secondary metabolites, it means that plant growth regulators and specific additives should be modulated to enhance *in vitro* production of biomass and secondary metabolites [71].

When nodal segments of *Ocimum basilicum* were cultured under the influence of different culture media including MS medium in different strengths and different combinations of PGRs, they expressed different values of methyl eugenol, linalool, and 1,8-cineole fractions [71]. Nodal segments are of *Cunila menthoides* medicinal plant cultured on MS medium containing different concentrations of PGRs resulting in biosynthesis of phenols, alkaloids, and terpenes in regenerated plants [71]. Media containing different types and concentrations of PGRs express different differentiation pathways and biomass values [72, 73], and it was associated with the expression of different types and concentrations of pharmaceuticals in cultured plant materials [74]. Contents of bioactive compounds in embryogenic callus and regenerated shoots of *Rosa rugosa* petal explants were influenced by PGRs type, concentration, and the

nitrogen source [75]. Also, in *Chonemorpha fragrance*, the amount of synthesized camptothecin was influenced by the PGRs type and concentrations [76].

The effect of carbon source concentration and type on culture biomass and metabolites productivity should be investigated. To enhance the biomass and biosynthesize of secondary metabolites, sucrose is widely used as a carbon source and it was better than maltose, glucose, and others [77]. During *in vitro* propagation, the optimal concentration of sucrose depends on plant species [15, 47, 68, 73]. For example, feeding the culture medium with 60 mM nitrogen and rise sucrose concentration from 3% sucrose to 5% increase the biomass production and camptothecin accumulation by 2.4-fold in the cell suspension cultures of *Nicotiana nimmoniana* [77]. In *Panax vietnamensis*, 2–5% sucrose enhanced the biomass and ginsenoside production in the cell suspension but 6–7% sucrose inhibited ginsenoside accumulation [78]. Geraniol production in transgenic tobacco cell suspension cultures was influenced by several culture conditions including carbon source light, and inoculums size [14].

Physical culture conditions can also affect the ability of *in vitro* cultured plants for the production of secondary metabolites. For example, light as one of these physical conditions can affect strongly on the production of secondary metabolites in *Abelmoschus esculentus* [79]. Culture media pH is an essential factor for the production of valuable plant material mass and its content of secondary metabolites. The optimum pH for normal plant tissue cultures is 5.8 but it should be changed if the purpose of the culture is to produce bioactive compounds. In a comparative study by Hagendoom et al. [80], on different plant species, they detected a positive correlation between acidification of the cytoplasm and the accumulation of different secondary metabolites including coniferin and lignin. In cell suspension of *C. roseus,* an increase in the pH of culture media between 4.3 and 9.0 was associated with a sharp increase in alkaloid production [81]. In general, low and high pH of the medium retard biomass and withanolide production in *Withania somnifera* cell culture [82], but the optimal pH was 4.5 for enhancing biomass production and Bacoside A formation [83].

In a scale-up production system, modulation the composition of the culture media is an essential prerequisite to enhance the production efficiency of a selected cell line, but long-term cultivation may lead to the reduction of the yield [64] due to an increase in somaclonal variation [84]. Consequently, genetic stability of the cultured plant materials should be established using determined indicators, such as molecular markers, stability of growth parameter index over extended subculture cycles, and metabolite production.

#### **5.2 Suspension and callus cultures**

Callus culture is an undifferentiated-unorganized mass obtained by cell division on cultured plant material on an agar medium. Then, calli are subcultured either for *in vitro* propagation through organogenesis or embryogenesis or used to establish suspension culture [85]. When callus in suitable texture is obtained on solid or semisolid agar medium and suspended in a specific liquid growth medium, the cells disperse and divide more and more producing cell suspensions. Then, cells can have faster and uniform growth rates associated with secondary metabolite production. Suspension cultures are the most widely employed PTC techniques in the production of secondary metabolites. When cells are grown in aqueous media to produce cell suspensions, some cells do not disperse in the medium and form tissue clumps, which disrupts growth and weakens the production of targeted secondary compounds.

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

Suspension cultures are also amenable for growth in small and giant fermenters, but these cultures may show genetic and biochemical variation. Under selected conditions, exploitation of cell cultures capable of producing medicinal compounds at a level similar or superior to that of intact plants.

Callus culture itself is exploited to produce and study secondary compounds in many medicinal plant species [66]. For induction of callus formation, specific culture conditions should be established, which means that cultured cells divide and proliferate rapidly as long as the cultural environment has sufficient nutrients and suitable growth regulators. Conditions for callus induction and proliferation are not favorable for the production of secondary metabolites. For induction of secondary metabolites, calli culture conditions should be changed or transferred to a new medium with a different composition [11]. High yields of proteolytic enzymes from the callus tissue culture of *Allium sativum* L. on MS medium containing NAA and BAP were obtained [86].

The advantages of the application of suspension-cell cultures are obvious including: (1) The biomass production is usually more rapid than that of other *in vitro* culture types as well as a whole plant, (2) Chemical and physical conditions can be easily controlled allowing the production of certain pharmaceuticals throughout the year if necessary, (3) Producers can provide their products in a sustainable manner that does not depend on large areas and leave the arable land areas to grow other crops, (4) The size and quality of the product can be controlled according to the market demand, (5) Producers can select plant cell line that ensures or improve product quality, and (6) Producers can combine more than one method, which leads to the development of new products.

Application of specific cell lines and selective culture of that cell lines lead to the production of secondary compounds more than those obtained from original tissues and normal culture conditions [87]. The addition of plant growth regulators enhances the production of target secondary metabolites in several medicinal plant species [88]. Cell immobilization [89] and genetic makeup [90] can be optimized to enhance the synthesis of secondary compounds under *in vitro* culture conditions. Cultured cells can be immobilized to form aggregates to enhance secondary metabolite production [91]. Cell immobilization is achieved through growing cells as aggregates or using substances such as alginate or polyurethane foam cubes [92].

Two-phase cell suspension cultures establish a growth medium for maximizing cell biomass and production of naphthoquinone pigment in the first phase, but the second phase was established at the dark condition and room temperature with alkaline pH. These two phases system enhanced biomass production six-fold and optimized metabolite production in *Arnebia* sp. [93]. In suspension cultures of *C. roseus*, cultures produced up to 20 g DW L − 1 of biomass. In addition, two phases culture technique increased active cell biomass with 10 times higher indole alkaloids production in comparison to that of the one-phase culture [94]. Under dark conditions at 25°C for 40 days, the two phases of co-culture of *Panax ginseng* and *Echiancea purpurea* adventitious root in bioreactors containing MS medium supplemented with IBA (25 μM), sucrose (50 g L − 1), and methyl jasmonate (200 μM) as elicitor for 30 days enhanced the production of ginsenosides and caffeic acid derivatives [95].
