Plant in vitro cultures represent an attractive and cost-effective alternative to classical approaches to plant secondary metabolite (PSM) production (the "Plant Cell Factory" concept). Among other advantages, they constitute the only sustainable and eco-friendly system to obtain complex chemical structures biosynthesized by rare or endangered plant species that resist domestication. For successful results, the biotechnological production of PSM requires an optimized system, for which

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... n has proved one of the most effective strategies. In plant cell cultures, an elicitor can be defined as a compound introduced in small concentrations to a living system to promote the biosynthesis of the target metabolite. Traditionally, elicitors have been classified in two types, abiotic or biotic, according to their chemical nature and exogenous or endogenous origin, and notably include yeast extract, methyl jasmonate, salicylic acid, vanadyl sulphate and chitosan. In this review, we summarize the enhancing effects of elicitors on the production of high-added value plant compounds such as taxanes, ginsenosides, aryltetralin lignans and other types of polyphenols, focusing particularly on the use of a new generation of elicitors such as coronatine and cyclodextrins. Molecules 2016, 21, 182 2 of 24 six years before they are ready for harvesting [3] , while Taxus trees reach a peak production of taxol only after 60 years of growth [4] . Consequently, even after considerable long-term planning, market demands for target compounds can be difficult to meet. Further, the extraction of fine chemicals from plants can be very challenging and expensive, giving poor yields. For all these reasons, in the last decades considerable effort has been invested in the biotechnological production of metabolites by means of plant cell and organ cultures. Plant cell factories constitute the most promising approach for a sustainable production of plant secondary metabolites of commercial interest [5, 6] , offering a continuous supply by means of large-scale culture. They have several advantages over the cultivation of medicinal and aromatic plants in the field [6]: (a) the desired product can be harvested anywhere in the world with strict control of production and quality; (b) independence from geographical or environmental fluctuations; (c) uncontaminated plant material is guaranteed, since plant cells are free of microorganisms, herbicides, pesticides and fungicides; (d) endangered plant species can be conserved for future generations; (e) growth cycles are of weeks rather than years as in the intact plant. Yet despite all these advantages, and the extensive effort invested in developing plant cell cultures as PSM production systems, commercially successful plant cell factories are still rare, which is at least partly due to a lack of knowledge of plant secondary metabolism and its in vitro control. The few industrially viable processes established to date produce pure compounds such as shikonin [7], taxol [8, 9] and berberine [10], or biomass, as in the case of ginseng roots [11] . An essential challenge for such a biotechnological system is to maintain costs below that of large-scale cultivation of plants. Given the complexity of in vitro cultivation techniques, together with the cost of sterilization and culture in bioreactors, new technologies are applied almost exclusively for the production of value-added compounds. The profitability of industrial PSM production depends largely on system productivity. In general, when cell or organ cultures are first established, yields are relatively low. Most attempts to increase biotechnological production have been based on an empirical approach, using a range of methodologies to enhance metabolite biosynthesis and accumulation. An alternative approach is to gain an understanding of the metabolic pathways involved. A protocol for the large-scale production of valuable secondary compounds first of all requires the establishment of callus biomass from selected highly productive plant genotypes. Calli, which are formed mainly by stem cells, have an unlimited growth capacity and can synthesize the same compounds as the original plant. Cell suspensions derived from the calli are first maintained on a small scale and then at bioreactor level. In all these steps, the culture conditions and environmental and physical factors (such as light, pH, temperature, shaking speed, etc.) need to be optimized to achieve a high production by assaying different culture media, hormonal combinations and carbon sources [12] . In the exponential growth phase of plant cell cultures, many metabolites are produced only at low levels, or not all, since their primary metabolite precursors are required for biomass formation. There is evidence that the induction of secondary metabolite production from primary compounds is more effective in the stationary growth phase. For this reason, a good strategy for a plant cell factory is to establish a two-stage culture, in which the cells are first maintained in an optimal medium for biomass formation and are then transferred to an optimal production medium that stimulates the synthesis of secondary compounds. This system has the advantage of allowing elicitors and biosynthetic precursors to be added at the time of maximum yield, that is, in the second phase of the culture [13, 14] . One of the most effective strategies for enhancing the biotechnological production of secondary compounds is elicitation. Although a cell culture can be elicited by physical factors, the addition of biotic or abiotic elicitors to the culture medium is the main methodology used in biotechnological cell cultures. Since it is impossible to consider the great variety of elicitors assayed in plant cell cultures in their entirety, in this review we have focused mainly on the action of the most commonly used and effective biotic elicitors for the biosynthesis and accumulation of secondary compounds of great interest for chemical-pharmaceutical industries. Molecules 2016, 21, 182 3 of 24 Elicitation Elicitation is one the most effective techniques currently used for improving the biotechnological production of secondary metabolites. Elicitors are compounds that stimulate any type of plant defense, promoting secondary metabolism to protect the cell and the whole plant [15] [16] [17] . According to their origin, elicitors can be divided into different types: (a) biotic and (b) abiotic. Abiotic elicitors can be considered as substances of non-biological origin, being predominantly inorganic compounds such as salts or physical factors [18, 19] . Inorganic chemicals like salts or metal ions have been used to increase the production of bioactive compounds by their modification of plant secondary metabolism. Salts (including AgNO 3 , AlCl 3 , CaCl 2 , CdCl 2 , CoCl 2 , CuCl 2 , HgCl 2 , KCl, MgSO 4 , NiSO 4 , VOSO 4 and Zn ions) can elicit PSM production in a variety of plant species in culture systems such as cell suspensions, hairy roots and adventitious roots [20] . In this review, however, we will be focusing on the more extensively explored biotic elicitors. The majority of biotic elicitors are recognized by specific receptors bound to the cell membrane. This stimulus is then transferred to the cell by a signal transduction system, producing changes that ultimately lead to the formation of phytoalexins [17] . The response of the plant is determined by several factors, principally its genetic characteristics and physiological state. In general, plant resistance to disease is controlled by plant resistance (R) and pathogen avirulence (Avr) genes [21] . However, while specific Avr products trigger defense responses in cultivars with matching R genes, the action of general elicitors can activate defenses in cultivars of more than one species [22] . According to the new concept of plant innate immunity, defense responses are triggered when plant cells recognize conserved microbe-associated molecular patterns (MAMPs), a new term for general and exogenous elicitors. Alternatively, a pathogen invasion can prompt the release of plant endogenous molecules/endogenous elicitors, termed as danger-associated molecular patterns. A second level of perception involves the recognition of pathogen-secreted effectors, formerly known as specific elicitors, which belong to different families, including proteins, glycans and lipids [23] .