**3. Culture systems**

phenylpropanoid, mevalonate, 2-C-methyl-d-erythritol-4-phosphate, amino acid, glucose, acetate-malonate pathway, or combined pathways. Secondary metabolites have an important role in the interaction between plants and their environment (e.g., defense against herbivores and pathogens, protection against ultraviolet light, etc.) and, thus, are vital for their existence and subsistence. They are accumulated in specific tissues and structures (e.g., vacuoles, specialized glands, trichomes, etc.), and their production is affected by several factors, like genotype, plant physiology, climate, environmental conditions, and pathogens;

Over the past decades, efforts have been directed at the extraction, structure elucidation, and evaluation of biological activity of many plant secondary metabolites. Plants continue to be the main source for many important bioactive molecules/pharmacophores [4, 5]. About 25–28% of modern medicines are derived from higher plants [6], and over 60% of anticancer drugs are directly or indirectly derived from plants [7]. According to a recent report of the British Broadcasting Corporation (BBC), plant-derived drugs will grow from \$29.3 billion in

In the last decades, considerable progress has been made concerning the production of secondary metabolites by using plant tissue culture techniques owing to the advantages of this platform over other production systems as discussed in the next section of this chapter. The most studied classes of plant secondary metabolites using plant cell and tissue culture production systems are alkaloids and the landmark example is the anticancer-registered drug Taxol® [3]. Plant tissue culture techniques were even endorsed by Food and Agriculture Organization as safe for the production of compounds for food application [9]. This chapter aims to discuss the main advantages of using plant cell and tissue culture techniques for the production of plant secondary metabolites as well as the different biotechnological approaches available to improve their production. Important and representative examples produced through these methods, as is the case of plant anticancer compounds and metabolites from *Lamiaceae* spp.,

**2. Advantages of plant tissue culture techniques for the production** 

In a context where consumers demand for safe natural products increases, because synthetic chemicals are perceived as potentially toxic, the interest in plant secondary metabolites from research and industry also increases [10]. Few important plant products with simple chemical structures can be produced via chemosynthesis; however, many compounds like alkaloids are difficult to be synthesized or the cost of their synthesis outweighs their commercial availability [11, 12]. Some compounds can be obtained from naturally grown plants, but sometimes there are regional and environmental restrictions, which can limit the commercial production [13]. Also, traditional cultivation of some plant species is difficult or takes several years. In this context, plant cell and tissue culture techniques appear as environmentally friendly alternative methods for the production of secondary metabolites when natural supply is limited

in some cases, they are only produced during certain developmental stages [2, 3].

2017 to around \$39.2 billion by 2022 with an annual growth rate of 5.9% [8].

are addressed.

**of secondary metabolites**

82 Secondary Metabolites - Sources and Applications

The production of secondary metabolites by *in vitro* cultures usually occurs in a two-step process, biomass accumulation and secondary metabolites synthesis, in which both steps need to be optimized independently [3, 14]. Production could be accomplished by using undifferentiated calli, cell suspension cultures, or organized structures like shoots, roots, or somatic embryos. In some cases, a certain degree of differentiation may be needed for the biosynthesis to occur [20]. The use of differentiated organ cultures is required, for instance, when the target metabolite is only produced in specialized plant tissues or glands as is the case of essential oils [20, 21].

Among differentiated tissues, hairy roots culture offers new opportunities for the *in vitro* production of plant-valuable compounds [22]. Hairy roots are induced by the infection of plants with *Agrobacterium rhizogenes*, a Gram-negative soil bacterium. During the infection, a DNA segment (T-DNA) from the large root-inducing (Ri) plasmid of the bacterium is transferred into the genome of the infected plant. The higher level of cellular differentiation, rapid growth, genetic and biochemical stability, and maintenance facility are some of the advantages of hairy roots [22]. Also, they can accumulate metabolites in the aerial parts of the plant. However, the difficulties in cultivating hairy roots in an industrial system limit their commercial use to produce valuable plant secondary metabolites.

and phosphate levels, plant growth regulators type and concentration, carbon source, etc. For instance, carbon source plays significant roles in the signal transduction systems through

Production of Plant Secondary Metabolites by Using Biotechnological Tools

http://dx.doi.org/10.5772/intechopen.76414

85

Secondary metabolites are produced by plant cells in response to environmental stimuli or as defensive mechanisms against invading pathogens. In this sense, the strategy available to improve the productivity of secondary metabolites, elicitation, aims to misguide the cells or tissues for a possible biotic/abiotic attack by using agents that trigger the defense response [33]. Elicitors have the ability to control an array of cellular activities at the biochemical and molecular level since they induce the upregulation of genes [33]. The elicitors can be biotic or abiotic and may comprise signaling molecules like methyl jasmonate, salicylic acid, microbial cell wall extracts (e.g., yeast extract, chitosan), inorganic salts, heavy metals, physical agents (e.g., UV radiation) among others [1, 34]. Methyl jasmonate and its related signal molecules, and salicylic acid are probably the most extensively used elicitors [5]. The combination of some elicitors with physical factors (e.g., UV light, temperature regime, and pulsed electric field) yielded good results for secondary metabolite production [35]. As reviewed by Giri and Zaheer [5], cell suspension culture is the most used culture system for elicitation treatment and secondary metabolites production. Due to its inherent characteristics of hormone autotrophy, uncontrolled growth, biosynthetic, and genetic stability distinctiveness, hairy root cultures have proved to be also a valuable culture system for elicitation experiments. In addition, there are some secondary metabolites that are synthesized only in the roots [14, 36, 37]. Multiple shoots culture is a less used culture system for elicitation treatments for the production of secondary metabolites which is particularly useful in the case of metabolites present in the leaves [5]. The elicitors can change the secondary metabolites production quantitatively and also qualitatively [5]. For extra information, consult the recent reviews on this subject [1, 5]. Nutrient and precursor feeding are also used to improve the yields of secondary metabolites production. Nutrient feeding involves the replenishment of nutrient medium, and in precursor feeding, plant cell cultures are used to convert precursors into products by utilizing preexisting enzyme systems [14]. Immobilization of plant cells is another strategy used to overcome problems of low shear resistance and cell aggregation. This procedure can be done by several methods, and the most widely used are surface immobilization or gel entrapment. In this technique, the cells are entrapped in a specific gel or a combination of gels. Examples of matrices used are calcium alginate (the most used), agarose, gelatin, carrageenan, or polyacrylamide [14]. This strategy has several advantages, such as the extension of cells' viability in the stationary stage, the simplification of downstream processing, the high-cell density within small bioreactors reducing the costs and risk of contamination, an increased product

regulating gene expression and developmental processes [3].

accumulation, the minimization of fluid viscosity, among others [38].

The permeabilization of plant cell membranes with chemicals, the use of electric field stress, and ultrasound techniques are strategies used to facilitate the removal of secondary metabolites from vacuoles and membrane systems of the plant cell, facilitating the secretion of prod-

The cultivated cells have the capacity for biotransformation of supplied compounds, which are not necessarily natural intermediaries of plant metabolism, into high-value compounds.

ucts into the culture medium and thus simplifying the purification process [14, 17].

Although there are many studies reporting the production of secondary metabolites using callus cultures and differentiated tissues [3, 14, 23], in most cases, undifferentiated cells are the preferred culture system [13]. Cell suspension culture is a simple and cost-effective method that has been extensively used to overcome the problems of large-scale production. Plant cell is biosynthetically totipotent, which means that under suitable conditions, each cell has theoretically the capacity to produce compounds identical to those present in the parent plant [13]. Plant cell cultures have more immediate potential for commercial application than tissue or organ cultures [21, 24]. They are considered as a stable system for the continuous production of secondary metabolites of uniform quality and yield. Another great advantage of plant cell cultures is the possibility to synthesize novel products not usually produced by the native plant [25, 26]. This is the preferable biotechnological platform to produce highvalue secondary metabolites, as taxol [27, 28], resveratrol [29], artemisinin [30], ginsenosides [31], and ajmalicine [32].
