**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

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

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

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

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.
