Application of Tissue Culture Techniques to Improve the Productivity of Medicinal Secondary Products from Medicinal Plants

Abstract

The plant kingdom is considered the most important source of medicinal chemicals. In vitro culture techniques are being considered a promising alternative to traditional agricultural processes to improve medicinal plants multiplication and their production of pharmaceutical compounds. In this chapter, several in vitro culture strategies are discussed to improve secondary metabolites production, including (1) plant kingdom as a source of medicinal chemicals, (2) in vitro culture of medicinal plants, (3) culture media optimization, (4) application of suspension cell culture for production of secondary metabolites, (5) elicitation to enhance the productivity of the culture, (6) precursor intermediates feeding, (7) selection of high-yielding cell lines, (8) overexpression of genes that control the production of bioactive compounds, and (9) scale-up production. Also, challenges that hinder the in vitro culture of medicinal plants using different techniques and the use of those techniques to produce pharmaceutical compounds are discussed in this chapter, including (a) secondary metabolites toxicity, (b) low growth rate, (c) culture browning, (d) limitation in the application of transformation, (e) somaclonal variation, and (f) vitrification. Therefore, the principal objective of the current chapter was to shed light on the studies on some medicinal plants and the used protocols to overcome some difficulties in terms of in vitro propagation that maximize their economic values.

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 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 disease-free 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 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. Vitrification-cryopreservation 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].

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].

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. 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].

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].

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 14^th 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 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 trans-activation 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 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 scaling-up 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. Semi-continuous 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].

6. limitations over secondary metabolite production in vitro

In general, there are many factors that may hinder the application of PTC for various purposes in the field of medicinal and other plant species. The production of medicinal compounds using PTC has two important aspects—the amount of plant materials should be sufficient for the production of the target substance, as well as the quantity and quality of the produced substance. Hence, it is necessary to identify and avoid the conditions and phenomena that may negatively affect the growth efficiency of the in vitro cultured plant tissue.

6.1 Avoidance of secondary metabolites toxicity

Obstacles facing the production of medicinal compounds from wild or cultivated plants can be avoided by using cell and tissue cultures, but these compounds may be toxic to the in vitro cultured cells or tissues and result in retardation of plant material growth and metabolite yield. Consequently, the toxicity of any secondary metabolite should be assessed and culture conditions should be modulated to avoid the production obstacles. On the other side, the toxic effect of a secondary metabolite can be beneficially used for the treatment of some illnesses, for example, cancer [166].

Long-term culture can be used for the accumulation of desirable metabolite(s), but it can be a problematic and limiting factor that should be avoided by the application of certain techniques, such as medium enrichment or substitution in bioreactors [167]. These strategies include accumulation of metabolites in vacuoles, and other subcellular compartments or the exudation of metabolites into the culture medium [168]. The last strategy needs the application of additional techniques to decrease the concentration of the accumulated metabolite leading to further biosynthesis. It is accomplished by changing the medium of the culture manually or mechanically. In this regard, hairy roots were recommended, but not all secondary compounds are synthesized and accumulated in the roots [66].

6.2 Avoidance of low growth rate of cultured plant materials

While successful production of a wide range of valuable secondary metabolites can be obtained using unorganized callus or suspension cultures, the differentiated organ can be used but each of them may face some problems. The most important problems are the slow growth rate and somaclonal variation [84]. Consequently, the production of secondary compounds through the application of PTC techniques becomes unstable at a specific period. Generally, the problems facing the production of secondary metabolites using PTC can be easily solved by changing the culture conditions to avoid growth retardation and somaclonal variation [11]. Also, the application of PTC techniques in combination with other approaches could be used to avoid growth retardation and genetic variation [11].

The appropriate conditions for increasing the growth of the cultured plant materials may be different from the conditions for increasing the concentration of the active substance. To overcome these dilemmas, a two-step protocol is used, one of which provides optimal conditions for growth and the other provides optimal conditions to produce the active substance [12]. For example, while growth stimulators should be used during the growth phase, elicitors should be used to stimulate the biosynthesis of active compounds [169].

Accumulation of secondary metabolites is obtained under the influence of biotic or abiotic stress, but it retards the biological mass. To ensure a high yield of secondary metabolites, producers hope to conserve conditions to stimulate high biomass and biosynthesis of the targeted metabolite. Consequently, optimization of culture conditions to increase growth parameters or application of elicitors become an essential prerequisite [169].

6.3 Avoidance of problems constrain the application of transformation in the production of active compounds

Despite Agrobacterium is an essential tool for gene transformation; sometimes some technical problems retard its application in some plants, it depends on genotype and/or transformation technique [170]. On the other hand, many factors can affect the efficiency of Agrobacterium-mediated transformation such as Agrobacterium’s optical density [171], antibiotic [118] or acetosyringone concentrations, and inoculation time [172]. All these difficulties should be avoided for the successful application of transformation techniques in the field of secondary metabolites production.

6.4 Avoidance culture browning

In vitro cultured explants release phenol compounds, which are oxidized by polyphenol oxidase and turned the media brown [173]. In woody plants, phenolic exudation appears early during the excision of plants causing browning of the cultured medium [173]. Browning closes the base of explants and retards the movement of nutrients from the medium into the cultured plant materials leading to retardation of plant growth. To overcome tissue browning, antioxidants or phenol absorbents, such as ascorbic acid, glutathione, activated charcoal, and polyvinylpyrrolidone were used. Also, transferring explants into new culture media at regular intervals can control the negative effects of the browning phenomenon [173]. To overcome the browning effect in the culture media in Glycyrrhiza inflata cell cultures, cultures were optimized in a bioreactor containing maximum cell concentration [174]. Dark conditions help to reduce the browning problem may be due to the reduction of the activity of the enzymes concerned with phenols synthesis and oxidation [175, 176].

6.5 Avoidance of somaclonal variation of the cultured plant materials

Production of the secondary metabolites using the cell culture technique is low during the early stage of growth where high carbon utilization exists and is associated with enhancement of primary metabolism. On the other hand, the production of secondary metabolites is high at the late stage when carbon is less needed for the production of primary metabolism [14]. Prolonged the age of the cultured plant materials is necessary but it may be associated with genetic variation [47, 84]. Therefore, the enhancement of growth criteria of the cultured plant materials is not sufficient to confirm the optimization of in vitro culture techniques for the production of secondary metabolites, but also genetic stability at the DNA level of the cultured plant materials is an essential parameter. For example, regenerates with high genetic fidelity and improved chemical profile of endangered C. spinosa L were reported, where the two-fold increase in flavonoids content than that of wild plants was obtained using methyl jasmonate and BAP [105]. Plant material with genetic fidelity after propagated in vitro culture was detected and used for the isolation of 20-hydroxyecdysone and polypodine B [177]. Production of true to type regenerants in Artemisia absinthium is very important in the commercial production of secondary metabolites [178].

Somaclonal variation results from chromosomal changes in number or structure, transposable elements, or possibly pre-existing genetic changes in the donor plant. To detect somaclonal variation, several molecular techniques such as Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR), and Simple Sequence Repeat (SSR) were recommended [18, 47].

6.6 Avoidance of vitrification

Sometimes, the production of secondary metabolites through some techniques such as cell suspension is not always an adequate procedure. Then, other techniques such as organ culture can be used as a supernumerary method for the production of secondary metabolites [85]. Shoot cultures as same as hairy root cultures are recommended for production of pharmaceuticals where they are genetically stable [179]. Shooty teratomas were produced for the production of secondary metabolites, such as vincristine in C. roseus [180] and naphthoquinone in Drosera capensis var. alba [181]. In some plant species, shoot culture showed vitrification problems, such as in moringa [47].

Generally, tissue culture plant materials were incubated in vessels to prevent microbial contamination and retard culture desiccation but these conditions may cause restriction of gases exchange between cultures and their surrounds. Under insufficient ventilation stress, the growth of the cultured plant materials was retarded due to retardation of photosynthesis, transpiration, and uptake of water and nutrients leading to the accumulation of ethylene and the appearance of vitrification or hyperhydricity [182]. The symptoms of vitrification are slowing growth rate, necrosis of shoot tips, loss of apical dominance, disorganized cell wall, fragile leaves, reduction of shoot multiplication, poor acclimatization, impaired stomatal function, reduction of some metabolites, alteration of ion composition, inhibition of H₂O₂ detoxification enzymes [183, 184].

Vitrification in medicinal and other plant species can be avoided by reducing the relative humidity and improving the aeration within culture vessels [183, 184], decreasing the concentration of free water by increasing the concentration of agar [185], and using anti-ethylene compounds including CoCl2, AgNO3 or salicylic acid [47, 183]. To confirm which anti-ethylene compounds can be used to conserve the genetic fidelity of in vitro cultured moringa shoots, fingerprinting profiles of the long-term culture (14 subcultures) were assessed using RAPD, SSR, and ISSR. While the application of silver nitrate improved plant multiplication and reduced vitrification but it resulted in higher somaclonal variation in comparison to salicylic acid [47].

7. Conclusion

An increase in the world’s population imposed an important matter, which is the inevitability of leaving arable land for food production. Where modern agricultural techniques can be used to produce secondary metabolites and preserve the genetic assets of these plants, the most notable technique is PTC. In addition, different PTC techniques are used to propagate rare and endangered plant species. Changes in the physical and chemical conditions of in vitro culture are easy and under control in a way that cannot be provided at all under field conditions. The ease of controlling the conditions of PTC conditions made it possible to use certain conditions to obtain true-to-type clones and their products, but other conditions are used to establish somaclonal variation for noval line selection.

The use of plant tissue techniques has become dependent on it to produce pharmaceutical materials after laboratory and applied experiments have proven that in vitro cultured plant materials are able to produce pharmaceuticals with the same amount and quality that can be obtained from soil cultivated plants. Moreover, the application of elite physical and chemical conditions of in vitro cultured plant materials made their production of secondary metabolites superior in quantity and quality to that of wild or cultivated plants. Therefore, to produce pharmaceutical compounds in large quantities to suit the increase in the population and increase their demand for safe medical products, tissue and cell culture techniques have been improved under several names including culture media optimization, the establishment of suspension and callus cultures, elicitation to enhance the productivity of in vitro cultures, application of precursor feeding as a substrate to improve the production of secondary metabolites, high yielding cell lines selection, enhance the overexpression of genes that control the production of bioactive compounds, application of genetic transformation using A. rhizogenes and application of “bioreactors” for scale-up production.

The use of PTC techniques to produce pharmaceutical compounds depends on the availability of production of sufficient-viable plant biomass to produce pharmaceutical substances with the requested quality and quantity. Therefore, it is necessary to understand all the factors that limit the production of targeted mass to avoid them such as the toxicity of secondary metabolites, low growth rate of cultured plant materials, and problems that constrain the application of transformation on a wide spectrum of plant species, somaclonal variation during cell or tissue cloning and verification of the cultured plant organs.