**3.5 Culture medium**

The tissue culture media are the basic support system for the growth and development of plant cell cultured in vitro. The activities of basic primary metabolism are largely influenced by the basal media considered to be common to most of the plant species. However, the differentiation or dedifferentiation of plant tissue cultures is influenced by the combinations of growth hormones mainly auxins and cytokinins (**Table 3**). The manipulation of media components have been reported to influence the biosynthesis and accumulation of secondary metabolites in plant cell cultures. Different strategies have been employed for improving secondary metabolite production in suspension cultures. The influence of media constituents and nutrient stress affect the production of diosgenin from callus cultures of *Dioscorea deltoidea*. The production of gentipicroside and swertiamarin was enhanced on MS medium supplemented with kinetin, NAA and 3% sucrose in suspension cultures of *Gentiana davidii* [44].

The productivity of picroside-1 was increased by optimizing the concentration of nutrients in growth medium and levels of phytohormones in the shoot cultures of *Picrorhiza kurroa* [45, 46]. Elevated sucrose levels from 3 to 6% were favourable in some cultures whereas addition of fructose promoted paclitaxel production in *Taxus* cell cultures [6]. Supplementation of MS medium with seaweed extract also contributed in enhancement of picroside accumulation in shoot cultures of *Picrorhiza* species [14].


#### **Table 3.**

*Pharmaceutical metabolites produced by hairy root cultures.*

*Production of Medicinal Compounds from Endangered and Commercially Important Medicinal… DOI: http://dx.doi.org/10.5772/intechopen.90742*

#### **3.6 Type and source of explant**

The type and source of explant has been of major importance in not only establishing successful tissue cultures in any plant species but also of significant importance in producing phytochemicals in vitro. The prime importance of choosing a right explant for the production of phytochemicals lies in the fact that the biosynthesis and accumulation of metabolites is very specific to tissues and organs along with their developmental stages. The tissue and developmental specific accumulation of phytochemicals thus makes it important that appropriate explant be selected for starting plant cell cultures for the production of phytochemicals.

Production of diosgenin has been carried out from cell suspension cultures of different explants of *Dioscorea doryophora* like stem-node, microtuber and intact tuber, etc., along with varying concentrations of sucrose in MS liquid media supplemented with 2 mg/L 2,4-D (0.3–3.5%). Increase in diosgenin production was obtained from tuber derived cell suspensions as compared to intact tuber explant.

Different cell lines were established on B5 medium supplemented with NAA by using stem- and needle-derived callus of *Taxus mairei* and taxol yield of upto 200 mg/L was obtained in precursor feeded cell suspensions [47].

#### **3.7 Light and temperature**

Plants tissue cultures are largely influenced by the quality and duration of light treatments. There are various case studies in the literature wherein manipulation of light parameters or the temperature regimes has resulted in the alteration in the production of secondary metabolites. Zhang et al. (2005) gave heat shocks of 35–50°C for 30–60 min in the suspension cultures of *Taxus yunnanensis* for enhancing the production of paclitaxel. Production was increased to six fold by pretreatment with abscisic acid. The production of swertiamarin and gentipicroside was enhanced in cell suspension cultures of *Gentiana davidii* by incubating at 25°C and light intensity of 2.33 Lux [44]. Increase in the concentration of glycyrrhizin was found in the root tissue of *Glycyrrhiza uralensis* grown under red light or under low and high intensity of UV-B radiations [48].

#### **3.8 Precursor feeding**

Exogenous supply of a biosynthetic precursor to culture medium also increases the yield of the desired products. The concept is based on the idea that any compound which is an intermediate, or is in the beginning of a secondary metabolite biosynthetic route, proves to be a good candidate for increasing the final yield of secondary metabolite. Varun et al. [49] has carried out exogenous feeding of immediate biosynthetic precursor, i.e., cinnamic acid and catalpol in the shoot cultures of *Picrorhiza kurroa* hence stimulated 4.2 fold production of picroside-1. The production of monoterpene alkaloids was increased in cell suspension cultures of *Catharanthus roseus* fed with precursor mevalonic acid, secologanin [50]. Callus cultures of *Dioscorea balcanica* fed with cholesterol, norflurazon as precursors were used for the production of diosgenin, phytosteroids [51]. Hallard et al. [52] used secologanin and tryptamine in cell suspension cultures of *Nicotiana tabaccum* for the production of strictosidine. Phenolics compounds were elicited from micropropagated plants of *Calligonum polygonoides* by Owis et al. [53].

Supplementation of media with amino acids has been found to enhance the production of indole alkaloids tropane alkaloid in cell suspension cultures [54, 55]. Addition of phenylalanine to cell suspension culture of *Salvia officinalis* enhanced

the production of rosmarinic acid. The production of taxol from *Taxus* cultures was also increased by using the same precursor [56]. Nicotinic acid was used as a precursor in the hairy root cultures of *Nicotiana rustica* for the production of nicotine. Hakkinen et al. [57] used hyoscyamine as a foreign substrate for enhancing the production of scopolamine in the hairy roots of *N. tabaccum* and found that 85% of the converted scopolamine was released into the medium.

### **3.9 Genotypic variation**

The biosynthesis and accumulation of secondary metabolites or phytochemicals of medicinal value is influenced by the genotype of the target plant species [58]. There are examples wherein genotypic variations have been reported for phytochemical content. However, there has been a technical problem in most of these studies because genotype collections are made from different locations, which vary in altitude, climatic conditions, etc. thus resulting in variation in accumulation of phytochemicals. It would be highly desirable and practically viable if the influence of genotypic variation on phytochemical content is investigated by collecting genotypes of a particular plant species and then growing under uniform environmental conditions. The variation for metabolite content can be done on those genotypic collections.

#### **3.10 Metabolic engineering for the production of phytopharmaceuticals**

True metabolic engineering of plant secondary metabolite pathways has been hampered due to the lack of thorough knowledge of biosynthetic pathways and their regulatory mechanisms leading to the formation of desired compounds. Methods like labeled precursor feeding, induced expression of regulatory genes and block competitive pathways and metabolism by antisense genes have been used for enhancing the production of desired metabolites. Yun et al. [59] cloned the hyoscyamine 6-beta hydroxylase gene (*h6h*) of *Hyoscyamus niger* and introduced into *Atropa belladonna* and collected scopolamine from engineered plant. In a later study, Hashimoto et al. (1993) reported fivefold higher concentration of scopolamine from *A. belladonna* hairy roots expressing the same gene than the wild-type hairy roots. Increased alkaloid production by overexpression of genes encoding key enzymes of tropane alkaloid biosynthesis pathway was reported by Palazon et al. [23] and Moyano et al. [60] in *Duboisia* hybrid, *Datura metel* and *Hyoscyamus muticis* hairy roots, respectively. Similarly, Zang et al. (2004) produced 411 mg/L scopolamine in cultivated hairy roots from the simultaneous over expression of *pmt* and *h6h* genes in *H. niger*. Elevated nicotine alkaloid production was achieved in *Nicotiana tabaccum* hairy roots carrying *h6h* gene [57]. Neha et al. [61] reported 2.6 fold increase in picroside-1 production by modulating four integrated secondary metabolic pathways, i.e., methyl erythritol phosphate, mevalonate, iridoid and phenylpropanoid pathway using seaweed extract. Moreover Sharma et al. [62] defined many strategies through metabolic engineering for stimulating the production of bio-active compounds from medicinal plants.

#### **3.11 Upscaling the production of phytochemicals**

The production of phytopharmaceuticals in cell cultures coupled with their low yield from natural sources and supply concerns of plant species has renewed interest in up scaling cell culture technology for large scale production. Bioreactors are the key step towards their commercial exploitation because it provides defined

*Production of Medicinal Compounds from Endangered and Commercially Important Medicinal… DOI: http://dx.doi.org/10.5772/intechopen.90742*

parameters for up scaling the production of phytochemicals or secondary metabolites from plant cell cultures.

Bioreactor is a large culture vessel fitted with microprocessor control unit for the control of pH, temperature, light, dissolved oxygen, gas flow rate, agitation speed, nutrient factors, cell density for optimal growth or production, handling of cultures, nutrient uptake and product harvestation, etc. The success of Mitsui Petrochemical Industry Co. Ltd. in Japan in producing shikonin on a commercial scale from *Lithospermum erythrorhizon* and that of Nitto Denko Corp. Japan in mass production of *Panax ginseng* or ginseng cells have demonstrated the practical feasibility of using cell cultures in the large scale production of secondary metabolites of pharmaceutical importance. Commercial companies like Phyton and Samyang Genex are successfully producing paclitaxel and its related taxanes on large scale [63].

Heble and Chadha (1985) reported the successful cultivation of *Catharanthus roseus* cells in 7–20 L capacity of airlift bioreactor for the production of ajmalicine and serpentine by judicious use of air lift and low agitation. Significant amounts of sanguinarine were produced in cell suspension cultures of *Papaver somniferum* using bioreactors [64]. Ginseng root tissue cultures in 20 ton bioreactor produced 500 mg/L of saponin per day [65]. Hahn et al. [66] have produced gensinoside from adventitious root culture of *Panax ginseng* through large scale bioreactor system. Chattopadhyay et al. [12] produced podophyllotoxin through cell cultures of *Podophyllum hexandrum* in a bioreactor.

Different types of culture systems have been successfully used such as airlift bioreactors were used for scaling up hairy root production of *Astragalus membranaceus* [67] and *Solanum chrysotricum* [68] and mist bioreactor for hairy root of *Tagetes patula* [69]. Flow diagram of a process for the production of picrosides from *Picrorhiza kurroa* is given below wherein callus cultures/suspension cultures have been established from different explants and accumulation of picrosides is being investigated by HPLC [46, 70] (**Figure 1**).

**Figure 1.** *Pictorial representation for picroside-1 production through plant tissue culture.*
