Metabolomics has developed into a major tool in functional genomics and plant systems biology. The various methods used for metabolomic analysis will be discussed from the analytical methods back to the preanalytical phase and the biological experiment. Particularly aspects of the preanalytical phase of the analysis is dealt with, including the risks of artefact formation with the various commonly used solvents. Metabolomics is like a snap shot, and conclusions from dynamic systems must be drawn with great care as demonstrated with a biosynthetic study of salicylate in Catharanthus roseus cell cultures.

Methods described in this paper are confined to in vitro dedifferentiated plant cell suspension cultures, which are convenient for the large-scale production of fine chemicals in bioreactors and for the study of cellular and molecular processes, as they offer the advantages of a simplified model system for the study of plants when compared with plants themselves or differentiated plant tissue cultures. The commonly used methods of initiation of a callus from a plant and subsequent steps from callus to cell suspension culture are presented in the protocol. This is followed by three different techniques for subculturing (by weighing cells, pipetting and pouring cell suspension) and four methods for growth measurement (fresh- and dry-weight cells, dissimilation curve and cell volume after sedimentation). The advantages and disadvantages of the methods are discussed. Finally, we provide a two-step (controlled rate) freezing technique also known as the slow (equilibrium) freezing method for long-term storage, which has been applied successfully to a wide range of plant cell suspension cultures.

As the only source for the low-abundance antitumor agents vinblastine and vincristine, Catharanthus roseus is highly valued and has been studied extensively as a model for medicinal plants improvement. The biosynthesis of these monoterpenoid indole alkaloids (MIAs) is a complex multistep enzymatic network that is tightly regulated by developmental and environmental factors. Here we review the knowledge achieved in the past 30 years of the MIA pathway in C. roseus, from genetic to metabolic aspects. Two early precursor pathways and a late mono-/bis-indole alkaloid pathway have been largely elucidated and established, as well as their intercellular and subcellular compartmentation. Many genes encoding constitutive structural biosynthetic enzymes, transcription factors, and transporters involved in these pathways have been cloned, characterized and applied in metabolic engineering strategies to improve the MIA production. However, genetic modification in the pathway in C. roseus resulted in complicated changes of both secondary and primary metabolism, affecting not only the MIA pathway but also other pathways. Research at metabolic level is required to increase the knowledge on the genetic regulation of the whole metabolic network connected to the MIA biosynthesis. Nuclear magnetic resonance-based metabolomics (metabolic profiling, fingerprinting and flux analyses) in combination with other ''omics'' have been implemented in studies of C. roseus for pathway elucidation, including among others, understanding stress response, cross talk between pathways, and diversion of carbon fluxes, with the aim to fully unravel the MIA biosynthesis, its regulation and the function of the alkaloids in the plant from a systems biology point of view.

In recent years, a new type of solvents, natural deep eutectic solvents (NADES), have been developed and are gaining popularity as an alternative "green solvent" to conventional organic solvents. The traditional extraction of flavor or fragrance from plant materials such as vanillin is a tedious procedure that involves the use of organic solvents. In this case the solubility of vanillin and its extractability from vanilla pods in 14 NADES was evaluated. The solubility in some NADES such as lactic acid: propanediol (620 mg/mL) and lactic acid: fructose (320 mg/ mL) was found to be almost similar to that of organic solvents e.g. methanol (632.94 mg/mL) or ethanol (375.81 mg/mL). Interestingly, the extractability of vanillin from vanilla pods did not reflect the results observed for the solubility tests. Some NADES with constituents that are suitable as food ingredients such as citric acid-fructose-glucose (CAFG), fructose-glucose (FG), malic acid-glucose (MAG), and malic acid-fructose-glucose (MAFG), showed a higher extraction power (7.6, 10.1, 16.7, and 16.3 mg/g DW, respectively) than ethanol (4.4 mg/g DW). The discrepancy between solubility and extractability might be due to differences in the interaction with the matrix depending on the NADES. The effect of water content in some NADES on the extractability of vanillin was also studied. With the high extraction capacity and the absence of toxicity of their components, NADES are very suitable for extraction of flavours and fragrances used in food and cosmetics. plants. 4 However, in terms of quality, natural vanilla has a superior aroma and fragrance performance than pure vanillin. This is due to the presence of various minor compounds in the pods that give special notes to the vanilla flavor. The highest quality vanilla extract is obtained from vanilla pods using the percolation or oleoresin method. 5The percolation method consists of circulating aqueous ethanol (35-50%, v/v) over the pods, under vacuum. This process takes betweenThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Besides alkaloids Catharanthus roseus produces a wide spectrum of phenolic compounds, this includes C6C1 compounds such as 2,3-dihydoxybenzoic acid, as well as phenylpropanoids such as cinnamic acid derivatives, flavonoids and anthocyanins. The occurrence of these compounds in C. roseus is reviewed as well as their biosynthesis and the regulation of the pathways. Both types of compounds compete with the indole alkaloid biosynthesis for chorismate, an important intermediate in plant metabolism. The biosynthesis C6C1 compounds is induced by biotic elicitors.