**10. Importance of modern biotechnologies secondary metabolites production**

The parts of plants to be used for therapy, nutrition, and other activities can be obtained from spontaneous or cultivated plants; the choice of production method is mostly determined by economic factors is affordable to collect spontaneous plants when abundant and costs are relatively low, however, in case of high collection costs and lack of spontaneous plants, cultivation can be less expensive [17]. Furthermore, a lot of spontaneous plants are collected without any control and are currently under extinction threat; just a small percentage is cultivated [104]—all these factors are of concern due to the decrease and loss of gene diversity and environmental degradation. Advantages of open field cultivations are related not only to the fact that they give a solution to a lack of vegetal material available in nature, but also to the fact that the wild plant often offers a highly heterogeneous which might be at the same time inadequate in terms of continuous supply and quality standards. Production of secondary metabolites from cell cultures is a valuable option for molecules that have elevated extraction costs and low output from plant material coming from cultivation [105, 106].

For these reasons and due to the current increased demand for natural food products and drugs of natural origin, the employment of biotechnological artificial culture systems might be a good alternative to conventional cultivations for *in vitro* production of secondary metabolites as well as a viable option to replace industrial biosynthesis products. These issues, together with the need to increase the production of plant materials with uniform quality standards, are encouraging pharmaceutical companies to innovate research aiming also at gene and cell technologies indicated as biotechnologies [107].

On one hand, *in vitro* cultivation systems give us the chance to exploit cells, tissues, organs, or organisms as a whole also through gene manipulation to obtain desired compounds [25]; on the other hand, they play a potential role in terms of large-scale productions, production from secondary metabolic pathways.

Plant tissue culture is based on the principle that the same substances found in nature inside an organ, a fruit, or other plant tissues can be induced to accumulate in undifferentiated cells while keeping gene information and the ability to produce that range of active principles detected in the mother plant [108].

Multiple factors influencing *in vitro* secondary metabolite synthesis can be found: type of raw material, environmental and climate conditions, culture media, the quantity of carbohydrates contained that influences biomass, type and quantity of hormones, light (optimal light quantity and intensity is a prerequisite for maximum expression of metabolites), temperature. Substrate composition strongly influences secondary metabolite production, especially for what concerns salt and growth regulators besides subsequent glucose addition that might increase accumulation if compared to cultures in which a fix concentration is used [109].

Plant cell cultures are defined also as "chemical factories for secondary metabolites" [25] and represent to date a viable alternative to the cultivation of pharmaceutical plants both from *in vitro* and non *in vitro* origin.

The most important reason for pharmaceutical companies to obtain valuable secondary metabolites in this way is due to the fact that conventional cultivations in fields of pharmaceutical plants of some species are time-consuming, expensive, and generate a reduced output.

Some large-scale protocols of productions for the market have been set up for extractions of berberine, shikonin, and *Ginseng* saponins [25, 109] by using bioreactors. Berberine is produced *in vitro* from two members of *Ranuncolaceae* (*Thalictrum minus* and *Coptis japonica*); shikonin is produced *in vitro* from *Lithospermum erythrorhizon* in quantities 800 times higher than quantities obtained from plant roots; saponins are produced *in vitro* from *Panax ginseng*.

Further research was performed on other secondary metabolites such as flavoring agents (i.e., vanillin produced in bioreactors from calluses explanted from *Vanilla planifolia* by the company ESCAgenetic Corporation—San Carlos—CA, USA), food coloring (e.g., anthocyanins from *Euphorbia milii*), drugs (e.g., taxol), different essential oils and natural insect repellents [25].

### **11. Conclusions**

Although for the production of food from plants there is an increasing tendency toward natural agriculture, in the production of substances intended for industry, in particular the medicinal industry, a cultivated or spontaneous plant cannot always guarantee a constant and high-quality product. Pollution problems, climate change, and the political unsafe of some harvesting and cultivation areas also make production uncertain. In this situation, the production of secondary metabolites *in vitro* ensures a safe and constant making of the substances of interest.

New technologies, always evolving, can give an even greater push toward *in vitro* culture, since they guarantee safe products, at lower costs, often difficult to obtain in nature.

#### **Acknowledgements**

The present work is co-financed by the RAS (Autonomous Region of Sardinia), under the Advanced Technologies for LANds management and Tools for Innovative Development of an EcoSustainable agriculture.
