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 elicitation 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.

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Elicitation, an Effective Strategy for the Biotechnological Production of Bioactive High-Added Value Compounds in Plant Cell Factories.

Dietary phytochemicals and their potential effects on obesity: a review.

There is an increased interest in secondary plant metabolites, such as polyphenols and carotenoids, due to their proposed health benefits. Much attention has focused on their bioavailability, a prerequisite for further physiological functions. As human studies are time consuming, costly, and restricted by ethical concerns, in vitro models for investigating the effects of digestion on these compounds have been developed and employed to predict their release from the food matrix, bioaccessibility, and assess changes in their profiles prior to absorption. Most typically, models simulate digestion in the oral cavity, the stomach, the small intestine, and, occasionally, the large intestine. A plethora of models have been reported, the choice mostly driven by the type of phytochemical studied, whether the purpose is screening or studying under close physiological conditions, and the availability of the model systems. Unfortunately, the diversity of model conditions has hampered the ability to compare results across different studies. For example, there is substantial variability in the time of digestion, concentrations of salts, enzymes, and bile acids used, pH, the inclusion of various digestion stages; and whether chosen conditions are static (with fixed concentrations of enzymes, bile salts, digesta, and so on) or dynamic (varying concentrations of these constituents). This review presents an overview of models that have been employed to study the digestion of both lipophilic and hydrophilic phytochemicals, comparing digestive conditions in vitro and in vivo and, finally, suggests a set of parameters for static models that resemble physiological conditions.

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In Vitro Models for Studying Secondary Plant Metabolite Digestion and Bioaccessibility

Improving production of plant secondary metabolites through biotic and abiotic elicitation

Higher plants undergo a variety of stresses and to combat those stresses they acclimatize themselves by producing diverse secondary metabolites. These secondary metabolites also have a wide range of industrial applications and hence they serve as candidates for commercialization. Owing to the constraints faced by natural plant extraction, plant cell/tissue culture has emerged as an alternative platform for the in vitro production of value added bioactive secondary metabolites. Implementation of several productivity enhancement strategies, including elicitation, can overcome the limitations faced by plant cell technology that hampers its extensive commercialization. Elicitation is a technique that involves exogenous addition of elicitors (abiotic or biotic) in the growth medium which consequently triggers stress response with concomitant enhancement in secondary metabolite production. Elicitor induced stress results in the activation of several defense-related genes or inactivation of non-defense-related genes, transient phosphorylation/dephosphorylation of proteins, expression of enzymes whose information can be used to ascertain the biosynthetic pathways of many secondary metabolites. Furthermore, integration of transcriptomics, proteomics and metabolomics with system biology can aid in discovery of novel genes, transcriptional factors and several biosynthetic pathways which in turn can serve as a valuable tool for metabolic engineering and gene manipulation for enhancing the yield and productivity of secondary metabolites.

Elicitation: a stimulation of stress in in vitro plant cell/tissue cultures for enhancement of secondary metabolite production

The effects of two elicitors: jasmonic acid and methyl jasmonate on cell growth as well as on rosmarinic acid accumulation in cell suspension cultures of Mentha × piperita were investigated. The highest rosmarinic acid accumulation 117.95 mg g−1 DW (12% DW) was measured 24 h after addition of 100 μM methyl jasmonate. A similar concentration 110.12 mg g−1 DW was detected 48 h after application of 200 μM jasmonic acid. Those values were nearly 1.5 times higher compared to the control sample, without elicitation. There was no substantial influence of elicitors on rosmarinic acid secretion into the culture media. Extracellular concentrations of rosmarinic acid were similar to the values from the control variants. It was documented that suspension cultures of M. piperita treated with elicitors showed a decrease in biomass accumulation when compared to the control.

The effects of jasmonic acid and methyl jasmonate on rosmarinic acid production in Mentha × piperita cell suspension cultures

This chapter is a comprehensive review of the health promotingphytochemicals commonly found in our daily food. These include carotenoids, phenolics, phytoestrogenes, polyunsaturated fatty acids, conjugated linoleic acids, tocols, allicin, glucosinolates, limonene and capsaicinoids. The review encompasses the main food sources of these chemicals in the diet, the possible mechanisms of their activity, evidence for potential health promoting activity and possible harmful effects. The newly emerged interest in these phytochemicals in animal nutrition as substitutes for synthetic antibiotic growth promoters has also been addressed.

Dietary phytochemicals and human health.

Nine polyphenols in the aerial parts of Mentha longifolia have been separated by chromatographic techniques. Their structures have been confirmed by HPLC/electrospray ionization-MS/MS. The compounds identified included rosmarinic acid, salvianolic acid L, dedihydro-salvianolic acid, luteolin-glucuronide, luteolin-diglucuronide, luteolin-glucopyranosyl-rhamnopyranoside, and eriodictyol-glucopyranosyl-rhamnopyranoside. The extracts of M. longifolia and M. piperita field plants, in vitro plants, callus tissues, and cell suspension cultures were profiled, and their polyphenol composition was compared in different tissues and quantified using ultra-performance column liquid chromatography (UPLC)/triple-quadrupole-MS in the selected-ion recording detection mode. Determination of desired compounds was based on calibration curves obtained for standards, which were previously isolated from M. longifolia aerial parts. The UPLC profiles revealed considerable differences in the synthesis of secondary metabolites among samples coming from field plants, in vitro plants, callus tissues, and cell suspension cultures. Plant tissues coming from field cultivation (for both M. piperita and M. longifolia) contained several phenolic compounds (flavonoids and phenolic acids), whereas plants from in vitro conditions, callus tissues, and suspension cultures contained only a few of them. Rosmarinic acid dominated in all of these samples. These results show that under in vitro conditions, the metabolism of phenolics undergoes a fundamental change.

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Determination of polyphenols in Mentha longifolia and M. piperita field-grown and in vitro plant samples using UPLC-TQ-MS.

The reaction to Tomato spotted wilt virus (TSWV) was evaluated in 94 accessions of Nicotiana, originating from the Institute of Soil Science and Plant Cultivation tobacco germplasm collection in Pulawy, Poland. Tests for resistance were conducted under greenhouse conditions using single TSWV isolate collected from tobacco plantation in Lublin district, Poland. The presence of the virus was verified using DAS-ELISA. SCAR markers associated with TSWV resistance gene were applied. The members of the section Alatae, the genus Nicotiana: N. alata, N. forgetiana, and Nicotiana x sanderae as well as N. tabacum cultivars: ‘Polalta’ and ‘Wiktoria’ with the TSWV resistance gene introduced from N. alata, displayed the hypersensitive reaction (HR) against TSWV (grade 0 on symptom intensity scale). In some of those accessions, the virus spread from the initially infected areas eliciting systemic hypersensitive reaction (SHR). Five accessions of N. alata and three of Nicotiana
 x
 sanderae were composed of 6.3–50.0 % of plants in which SHR symptoms appeared. In all of N. forgetiana plants HR reaction was followed by systemic infection (SHR). In N. tabacum ‘Wiktoria’ 21.1 % of plants showed HR reaction, while the remaining were susceptible (S). All of the genotypes which responded with HR or SHR reaction to TSWV infection demonstrated the presence of SCAR markers linked to the resistance gene. The remaining eighty tested accessions were identified as being susceptible upon exposure to TSWV.

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A survey of Nicotiana germplasm for resistance to Tomato spotted wilt virus (TSWV)

Growing resistant cultivars is the best method of protecting the crops against Potato virus Y (PVY). There are a few sources of PVY resistance/tolerance in tobacco acquired through mass selection, X-ray induced mutagenesis and introgressions from wild Nicotiana species. Here, we compare major sources of PVY resistance/tolerance in inoculation tests using ten PVY isolates collected in Central Europe (Poland and Germany) and differing with their virulence. The diversity of collected isolates was confirmed by DAS-ELISA tests and two PCR assays targeting the most common recombination sites in the PVY genome. We used these isolates in inoculation tests on five resistant cultivars 'V.SCR', 'PBD6', 'TN86', 'VAM', 'Wiślica', a tolerant breeding line 'BPA' and four susceptible cultivars 'BP-210', 'K326', 'NC95', 'Samsun H'. None of the tested cultivars/breeding lines showed universal resistance against all ten isolates. However, 'VAM' and 'Wiślica' appeared to be the most effective sources, as they showed no symptoms and gave negative DAS-ELISA tests for four out of ten tested PVY isolates. In contrast, tolerance of the breeding line 'BPA' was effective against all tested isolates, because inoculation did not lead to development of full disease symptoms in that breeding line.

Anna Czubacka

Papers

The effects of jasmonic acid and methyl jasmonate on rosmarinic acid production in Mentha × piperita cell suspension cultures

Dietary phytochemicals and human health.

Determination of polyphenols in Mentha longifolia and M. piperita field-grown and in vitro plant samples using UPLC-TQ-MS.

A survey of Nicotiana germplasm for resistance to Tomato spotted wilt virus (TSWV)

Resistance vs. tolerance to Potato virus Y in tobacco-comparing effectiveness using virus isolates from Central Europe.