**1. Introduction**

Unlike animals, plants do not have the ability to move, making them vulnerable to attack by pests and sometimes animals. To overcome this problem, plant tissues synthesize enormous compounds, such as terpenes, polyphenols, cardenolides, steroids,

alkaloids, and glycosides, and use them as defense strategies [1]. These defense compounds are called secondary metabolites and are not necessary for essential plant functions, such as growth, photosynthesis, and reproduction. These compounds are accumulated in the plant body to use by man as pharmaceutical, agrochemicals, aromatics, and food additives [1, 2]. Despite the progress in synthetic chemistry, plants are considered the most successful sources of drugs due to their bioactive compounds produced through secondary metabolism pathways [2].

In industrialized and developing countries, raw plant materials and plant-derived pharmaceuticals have naturally an essential component of present-day human healthcare systems. A known fact is that over 80% of the human beans use herbal medicines for healthy living [3]. In this respect, at present, more than 40% of the used pharmaceuticals by Western countries are derivatives of natural resources [4]. Worldwide, man uses about 35000–70000 plant species to prevent and cure diseases, most of them are reported in China (10,000–11,250), India (7500), Mexico (2237), and others [5]. Quality assurance and standardization of herbal medicines during the collection, handling, processing, and production of herbal medicine are essential prerequisites to ensure safety for the global herbal market. Wild plant materials are collected from gardens, open pasture, or forest land. In some cases, medicinal plants grow like weeds on agricultural land. While the bulk of the medicinal plant materials is still wild-harvested, a very small number of plant species are cultivated commercially [6]. However, increase populations and urban growth were associated with an over-exploitation of natural resources. Unfortunately, several medicinal plant species are disappeared due to the expansion of land for the purpose of growing crops, urban expansion, uncontrolled deforestation, and intensive collection [7]. Now, the increase in demand for these compounds encouraged the cultivation of large areas of medicinal plants and the application of new technologies, such as plant tissue culture (PTC) to preserve them from extinction and improve their productivity in quality and quantity.

Manufacturing of medicinal products from soil-grown plants faces some challenges, such as: (1) The wild-targeted plant does not exist in sufficient abundance in the local environment or is rare in general, (2) Cultivation of the target plant may need certain conditions, (3) Production of the target substance may require to grow plants for a long time, (4) The target substance may present at low concentration in cultivated or harvested plants, (5) Variations in environmental conditions may result in the production of bioactive compounds at a non-homogeneous quantity or quality, (6) Collection of plants for pharmaceuticals may be unsafe, (7) Harvest of propagated medicinal plants for drug industries is time- and money-consuming [8]. To overcome all the obstacles, PTC techniques express the great potential for bioproduction of phytoconstituents of high therapeutic value. By application of artificial techniques, regulation of the biosynthetic pathway of the certain plant to enhance the production of valuable compounds or avoidance of production of an unwanted substance become possible.

With the aid of gene technology and molecular techniques, *in vitro* culture procedures, such as cell, organ or tissue culture, somatic embryogenesis, somatic hybridization, genetic transformation, hairy roots, and induction of somaclonal variation, and others can be applied to the improvement of bioactive compounds yields. For example, recombinant DNA technology can be used to direct metabolic pathways and produce pharmaceuticals, such as antibodies and hormones. These *in vitro* culture techniques are better than others where they are carried out under precisely controlled physical and chemical conditions. PTC techniques are a resolution for the propagation of seedless medicinal plants and others with small or unviable seeds that *DOI: http://dx.doi.org/10.5772/intechopen.105193 Application of Tissue Culture Techniques to Improve the Productivity of Medicinal Secondary...*

not be able to germinate in soil [9, 10]. In addition, PTC techniques hold significant promise for true to type, disease-free, rapid and mass multiplication, and plant development [11].

Application of PTC technologies in the medicinal plant does not free from problems but avoiding their problems can be precisely controlled, which makes *in vitro* cultivation an ideal alternative to produce medicinal compounds from plants [12]. One of the obstacles is that the prices of the products resulting from biotechnology are higher than other products resulting from cultivated or wild plants. In this concern, the application of large-scale PTC techniques have been found to be an attractive alternative tools to the traditional plantations, where they offer a controlled supply of secondary metabolites independent of plant availability and a more consistent product in quantity and quality [13]. In the last decade, to meet pharmaceutical industry demand and conserve natural sources, researchers concentrated their efforts on optimizing culture conditions for maximizing the obtained yield of targeted secondary metabolites by application of several artificial-developed techniques [14].

Through PTC techniques, a whole plant can be regenerated from an organ, small tissue, or a plant cell but it should carry out on a suitable culture medium and under a controlled environment [15]. Under these conditions, the obtained plantlets are true to type and show characteristics identical to the mother plant. On the other hand, the culture conditions can be controlled to stimulate genetic variation for plant improvement, but it requires the construction of a selection procedure to select an elite mutant. For several decades, *in vitro* culture techniques are being used increasingly as a supplement to traditional breeding tools for the modification and improvement of plants. For example, *Coryodalis yanhusuo*, an important medicinal plant was improved through the application of the somatic embryogenesis technique to produce diseasefree lines [16]. While PTC can be established from any part of a plant, meristematic tissues, such as shoot tip or nodal segments, are usually recommended [15, 17, 18]. In addition, the physiological state of the donor plant affects strongly on regeneration ability of the cultured plant materials [9, 18].

The application of PTC techniques in the medicinal and other plant species becomes an essential prerequisite for plant propagation and improvement [15, 17]. The application of plant tissue culture has several advantages: (1) It results in the production of thousands of plantlets in a short period from a small segment of the tested plant. (2) It is a main procedure to obtain pathogen-free plants. (3) It can be used to culture plants round the year, irrespective of weather or season. (4) It needs little space for the propagation of the southlands of plants. (5) It can be used as the main procedure to produce a new cultivar of a certain plant. (6) It can be used to understand the effect of a specific biotic or abiotic factor on a tested plant beyond the interaction of other factors. (7) It helps to understand the molecular biology of plant differentiation. (8) It is an essential prerequisite during the production of genetically engineered plants. (9) It is an effective procedure for the production of pharmaceutical compounds. (10) It is an essential procedure for the preservation of endangered plant species, genetic assets, and gene banks.

### **2. Plant kingdom as a source of medicinal chemicals**

Phytotherapy becomes a complementary and important part of pharmacotherapy and modern medicine. It is a type of treatment based on natural medicinal resources (drugs) and herbal remedies for the purposes of prevention and treatment of illness.

Herbal drugs mean using the whole plant or part of it, fresh or dry, to treat or prevent human disease. Any plant part (flower, leaf, root, bark, fruit, and seed), resins, balsams, rubber, plant exudates, algae, fungi, or lichen can be used as herbal drugs for its medicinal properties. Herbal drugs or herbal remedies contain active ingredients of herbal medicinal products. The aerial plant parts, such as leaves, seeds, and flowers, are often able to synthesize and accumulate secondary metabolites more than those obtained by underground parts, such as roots or rhizomes [19]. For example, in *Scrophularia kakudensis*, the total phenol and flavonoid, as well as free radical scavenging compounds, were higher in shoot than root extract [20]. The variable contents of bioactive compounds in different plant tissues may be due to the specialized ability of each tissue to synthesize the bioactive ingredients or their ability to store them considering the physiological condition and endogenous hormone levels [19].

Based on their biosynthetic origins, reports classify the bioactive secondary metabolites of the plant into major groups, including phenolic compounds, terpenoids, nitrogen-containing alkaloids, and sulfur-containing compounds [21]. Phenolic compounds were the most important group where they are largely used to enhance human health and they naturally occur in fruits, vegetables, cereals, and beverages. Phenols are classified into different groups, including phenolic acids, flavonoids, stilbenes, and lignans, and they include apigenin, diosmin, quercetin, kaempferol, eriodictyol, naringenin, hesperetin, baicalein, chrysin, catechin, morin, genistein, curcumin, colchicine, resveratrol, and emodin. For the production and extraction of hundreds of these secondary products, plant cell, tissue, or organ cultures were used [21].

As a part of complementary and alternative medicine, medicinal plant extracts are widely used in chronic diseases like diabetes, hypertension, cancer, etc. Melatonin and serotonin, as antioxidants, were detected in the field and greenhouse-grown *Ocimum sanctum* L. plants [22]. Extract of *in vitro* cultures of *Hovenia dulcis* has antitumor effects [23]. *Aegle marmelos* can be used as antibacterial, antifungal, antidiabetic, and antioxidant [24]; it is also useful to treat several symptoms, such as stomachalgia, diarrhea, dysentery, malaria, and fever [25]. *In vitro* propagated *Artemisia japonica* was used to obtain antioxidant, insecticidal, antimalarial, antisporulant, antimicrobial, cytotoxic, and osteoinductive activities [26]. Acacetin (5,7-dihydroxy-4-methoxyflavone) has several therapeutic effects, it is found in more than 200 plant species belonging to 60 plant families especially *Asteraceae and Lamiaceae* families [27]. Acacetin is used for antiplasmodial, anticancerous, antidiabetic, antiperoxidative, antipyretic, anti-inflammatory, and antiproliferative activities [27]. Several compounds with anti-uveal melanoma activity were extracted from *Acacia nilotica,* including gallocatechin 5-O-gallate, methyl gallate, gallic acid, catechin 5-O-gallate, catechin, 1-O-galloyl-β-D-glucose, digallic acid, and 1,6-di-O-galloyl-β-D-glucose [28]. Biotechnological systems can be used to obtain vaccines from many plant species to provide immune protection against diseases [29]. Production of plant-based edible vaccines is mainly manipulated by the integration of the transgene into *in vitro* cultured plant cells to produce the antigen protein for specific diseases [30].

Screening of 346 methanol extracts of 281 native and cultivated plant species in Egypt indicated that *Agave americana, A. lophantha, Furcraea selloa, Calotropis procera, Pergularia tomentosa, Asclepias sinaica, Alkanna orientalis, Khaya grandifoliola, Swietenia mahogani, Pimenta racemosa, Pinus canariensis, Verbascum sinaiticum, Solanum elaeagnifolium, S. nigrum, and Brachychiton rupestris* have strong antischistosomal activity [31]. In addition, the antioxidant activity of the extract of 90 plants *DOI: http://dx.doi.org/10.5772/intechopen.105193 Application of Tissue Culture Techniques to Improve the Productivity of Medicinal Secondary...*

was determined by 2, 2 diphenyl-1-picrylhydrazyl (DPPH) assay [32], and extracts of some plant species expressed high antioxidant and cytotoxic activities that inhibited the growth of cancer cells [33]. Leaves of *A. marmelos* contain several medicinal compounds including π-sitosterol, lupeol, aegelin, rutin, flavone, glycoside, marmesinine, oisopentenyl halfordiol, phenylethyl cinnamides, and marmeline [24].

### **3. Application of** *in vitro* **culture techniques on medicinal plants**

Plant tissue culture is the most promising savior of medicinal plants that face problems of low yield and susceptibility to biotic or abiotic stress. Also, PTC can be used for *in situ* and *ex situ* conservation, propagation, polyploidy or aneuploidy induction, plant engineering, and bioreactor applications. *In vitro* multiplication was established in many threatened and endemic medicinal plants, such as *Bacopa monnieri* [34], *Paedaria foetida* [35], *Picrorhiza kuroa* [36], *Salvadora persica* [37], *Potentilla fulgens* [38], *Eryngium foetidum* [39], and *H. dulcis* [40].

High multiplication using seedling tissues or shoot meristems was achieved in several plant species, such as *Citrullus colocynthis* [41], *Zephyranthes bulbous* [42]*, Plectranthus vetiveroids* [43], *Glossocardia bosvallea* [44], *Cannabis sativa* [45], *O. sanctum* L. [22], *Caralluma retrospeciens* [46], *Solanum nigrum* [15], *Moringa oliefera* [47], *Pulicaria incisa* [48], *Rosa damascena* [49], *A. marmelos* [50], *Artemisia judaica* [51], and *Hyoscyamus muticus* [52].

For long-term storage of medicinal plant materials, cryopreservation is recommended where it is carried out in liquid nitrogen (−196°C). Different plant organs or parts, including seeds, corms, bulbs, rhizomes, roots, tubers, buds, and cuttings, can be stored for conservation purposes [11], especially in medicinal plants with recalcitrant seeds. The main applied techniques of cryopreservation of medicinal plants are vitrification, desiccation, and encapsulation–dehydration. Vitrificationcryopreservation of shoot tips of *Dioscorea floribunda* medicinal plant indicated that the genome of cryopreserved shoot tips was stable upon application of molecular, morphological, and biochemical procedures [53]. Vitrification–encapsulation–dehydration techniques of *Dioscorea deltoidei* medicinal plant shoot tips proved that the secondary metabolites of cryopreserved shoot tips were like control plants [54].

PTC is more efficient than naturally grown plant materials to assess the effect of different experimental conditions on the production of secondary metabolites of medicinal plants [55]. PTC opens the way for the production of engineered molecules and produces new forms of plant secondary metabolites [56]. These new forms of compounds may have a valuable effect on biological control, food, pharmaceutical, and other strategies. Transformation techniques are widely dependent on PTC for enhancing the *in vitro* production of valuable plant secondary metabolites [57].

Different types of PTC techniques are successfully exploited for *in vitro* propagation as well as synthesis and extraction of secondary metabolites [12]. Sometimes root culture is recommended because it provides valuable biomass in a short time and stable metabolite productivity. In addition, root cultures express genetic stability for long-term culture compared to other forms of *in vitro* cultures, such as cell aggregates and rhizoids. Roots are fully organized plant organ, ensures biochemical stability, and usually express the full biosynthetic capacity as same as soil-grown plant root. *In vitro* root cultures could be a better alternative for the accumulation of elevated contents of secondary metabolites. For example, root cultures of *Hemidesmus indicus* were used as a tool for *in vitro* production of 2-hydroxy 4-methoxy benzaldehyde [11, 58].

*In vitro*-produced hairy roots are formed without connection with any other plant organs. Then, the synthesized metabolites are not transported to other plant parts and are accumulated where they are synthesized. The produced secondary metabolites may be present in minor, undetectable quantities *in vivo* but they are present in higher levels in hairy roots due to the optimized culture conditions (14). Consequently, mass production of secondary compounds in the bioreactor was established using hairy root cultures [59].
