**6. Growth curve**

In cell suspension, depending on environmental parameters and on bioreactor features, the development of cells cultured on the liquid substrate is based on specific phases illustrated in **Figure 5**. The graph shows time (horizontal axis) and cell number (vertical axis). At the beginning a slow-growth phase is shown, known as lag phase followed by a phase in which cell concentration grows based on a logarithmic scale,

**Figure 5.** *Growth curve of a cell suspension culture.*

log phase; then a second slow-growth phase occurs followed by a phase in which the culture is numerically constant indicated as a plateau or steady state.

During the latency phase, reduced growth and an accumulation of substances useful for cell development occur, while during the exponential growth phase a considerable biomass increase can be observed. In a discontinue culture, in the case that cells accumulate metabolites in vacuoles, the biomass is removed at the end of the exponential phase; during the stationary phase a balance occurs between new cells and dead cells, then secondary metabolites are excreted in culture media. In this case, the collection is carried out by replacing from time to time or continuously the culture media.

#### **7. Production increase**

In the production of high-value secondary metabolites, a good strategy is offered by the use of technologies that ensure elevated yield and stable over time. It should be underlined that the production of secondary metabolites from plants is genotypedependent and this fact influences both metabolite type and quantity. Mother plants can be selected to a first selection to identify plants that ensure also *in vivo* a higher production of compound needed. Once the *in vitro* culture is stabilized, both from cells and organs, hyperproductive lines can be selected [23]. Selection is carried out through *in vitro* growth analysis on cell lines or organs, evaluating the multiplication degree but also assessing the quantity and quality of metabolite produced through chromatography and spectroscopic analysis [23].

The output can also be increased through conventional systems or metabolic engineering methodologies [22, 55].

#### **7.1 Biosynthesis pathways**

By using metabolic engineering, the biosynthesis pathways can be studied more efficiently [56, 57] through studying gene overexpression that alterates pathways. The study design includes analysis of enzyme reactions and biosynthesis processes at genetic, transcriptomic, and proteomic levels; in addition, it is also studied the manipulation of genes that encode critical enzymes and those that regulate the speed in the biosynthesis pathways [58, 59]. However, to date this system is limited to experimental settings and no method has been identified yet for industrial transfer of such methodology. Currently, the study of the biosynthesis pathway in phenylpropanol seems to be one of the most promising given that this substance is involved in the biosynthesis of different secondary metabolites in plants [60, 61].

#### **8. Conventional technologies**

Culture parameters are among the factors that mostly influence secondary metabolite production—substrate composition both in terms of mineral and organic compounds; pH; characteristics of cell inoculation; physical parameters, such as temperature, light intensity, duration, shaking, and aeration [22, 23, 27]. The substrate should be selected based on the requirements of plant species. Each substrate parameter can be modified to better adjust to the species and to metabolites to be obtained by it—salt type and concentration, carbon source, growth regulators. In nature,

#### In Vitro *Cultures for the Production of Secondary Metabolites DOI: http://dx.doi.org/10.5772/intechopen.101880*

secondary metabolites production is in response to environmental stimuli, or for defensive purposes. This mechanism can be simulated in the laboratory through the modification of the culture parameters, for example, light, temperature, or through the use of substances called elicitors. To elicitors belong both organic and inorganic molecules, such as 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 (**Tables 2** and **3**).

Aid to the production of new secondary metabolites, or increased production of those already known and used, can come from new technologies, such as transgenic



#### **Table 2.** *Abiotic elicitors.*

cultures. Several works have demonstrated the safety of these technologies, and their effectiveness, at low cost, for the production of secondary metabolites for medicine and industry [103].


**Table 3.** *Biotic elicitors.*

### **9. Potential applications in agriculture**

Focusing on biodiversity can be useful to strengthen food security and human nutrition aiming at promoting general sustainable development. Traditional crops represent an important biodiversity source and carry out a key role in preserving the identity of specific production areas as well the consumer behavior and transfer of cultural heritage to next generations. However, these cultures and foods require to be preserved from genetic erosion that can determine tragic effects on biodiversity, environmental sustainability, and rural economies.

As a matter of fact, this methodology based exclusively on a phenotypic evaluation does not allow to easily distinguish between genotype and effects on the environment. Recent methodologies based on gene markers enable us to identify species, cultivars, and autochthone varieties easily and rapidly.

Elevated costs and technical problems that might arise when the relationship between phenotype features and gene expression is studied, make the application of these methodologies often difficult. Recently, secondary metabolite analysis has been proposed as a crucial tool to identify a specific species; the metabolic profile, in fact, can lead to the identification of a huge quantity of local autochthone varieties, acting against globalization of agriculture production and being at the same time a tool to identify metabolites useful in traditional project characterization.
