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The Action will be in compliance with the general rules of the COST Framework and will include: (i) -State-of-the-art conferences, workshops and seminars. (ii) Exchange of experts, scientists, and graduate students for training, especially in the form of short-term scientific missions, and (iii) Exchange of reports, publications, and experimental procedures.
The scientific/technological areas will be those focusing on development of OMICS technologies for the
-Characterisation of Saffron genetic resources and crop breeding, including genomics, transcriptomics, epigenomics, proteomics, metabolomics, interactomics, cytomics and bioinformatics. The following issues are being approached initially (i) high resolution mass spectrometry for the characterisation of metabolites found in Saffron stigmas; (ii) massively parallel sequencing of the transcriptome of Saffron stigmas, for the identification of transcripts responsible for the synthesis of key metabolites, (iii) alignment of transcriptome sequences from C. sativus, and from its putative ancestors to verify the auto-, vs. alloploid and the mono- vs polyphyletic origin of this species, and (iv) SNP mining of the transcriptome sequences of different ecotypes of C. sativus, with the aim of uncovering intraspecific variability; development of SNP assays useful for the non-ambiguous identification of local ecotypes.
-Development of techniques to detect new-generation biological adulterants. Briefly, pure Saffron is composed exclusively of dried stigmas of Crocus sativus L. flower, a perennial bulbous. The high price of saffron makes it frequent subject of adulteration of various types, including the mix of colorants and authentic Saffron stigmas with flowers and other parts of different plants artificially coloured with organic dyes (tartrazine, Sudan red, etc.), that can be detected by suitable analytical techniques (ISO 3632) . Recently, however, massive adulteration of Saffron with natural dyes has been detected. The chemical composition is almost identical to that of Saffron, rich in crocin and crocetin, typical carotenoids of C. sativus stigmas. This chemical similarity means that quality control standard techniques fail to detect such adulteration. The commonly called 'Gardenia Yellow' and 'Buddleja Yellow' are extracts from the fruits of two plants, Gardenia jasminoides Ellis. and Buddleja officinalis Maxim., respectively, which are sold as natural dyes from China at low price, and even through the Internet (e.g. www.herbalextractonline.com/Natural-Food-Coloring/). With such colorants and staining various filamentous materials, it is possible to create cheap "pseudo-Saffron", with good colorant strength and chemical composition similar to Saffron, but without the organoleptic characteristics of the real spice. Alone or used to "cut" the real Saffron spice we face a new refined form of adulteration that, of course, must be prosecuted, because endangers the honourable Saffron production and the product's reputation. To date, detection of this last-generation biological adulteration requires the use of sophisticated chemical analysis techniques (HPLC, LC-MS-MS and LC-DAD). Despite their obvious usefulness they have the disadvantage of high cost in equipment, personnel and time, as well requiring large amounts of sample (in the order of milligrams) and, sometimes, previous extractions. Modern genetic fingerprinting techniques (genomics), imported from forensic science, is an alternative technology. Among its main advantages are that are highly specific, require very small amount of sample for analysis (micrograms) and require no preparative extractions. Therefore, the genetic fingerprint is considered an ideal technology to control the purity of food products. There is only one scientific paper that establishes a molecular method for detection of foreign plant material in the samples of saffron spice [Ma XQ, Zhu DY, Li SP, Dong TT, Tsim KW (2001) Authentic identification of stigma Croci (stigma of Crocus sativus) from its adulterants by molecular genetic analysis. Planta Med. 2001 67:183-6]. These authors sequenced the ribosomal RNA spacer regions of C. sativus, and three species that are frequently used in the adulteration of saffron in the Far East, Carthamus tinctorius L., Hemerocallis fulva L. and Hemerocallis citrina Baroni, finding multiple polymorphisms among these sequences that could be used for the design of molecular markers of adulteration. However, no records of work designed to detect massive adulteration with Gardenia and Buddleja.
-Implementation of traceability tools. Fraud involving the origin of saffron is widely detected. Several Protected Denominations of Origin (PDOs) are established in Europe (see C5). The purpose of these protected labels is to preserve cultivation, manufacturing and merchandising of Saffron in their specific areas. Excellence and gourmet quality of Saffron has always been associated to European productions. Although major production comes from third countries: Iran (Khorasan), Morocco (Taliouine –Montagnes du Sirwa- & Vallée de L’Ourika), and India (Kashmir), as Saffron in bulk, the spice is manufactured, packaged and commercialised in European brands. To differentiate Saffron of different origins (areas of cultivation and/or manufacturing) based on their physical-chemical or organoleptic features is not easy. Unfortunately mixtures (as blends or coupages) of expensive European Saffron with cheap Saffron mainly from Iran are very common nowadays. These mixtures are marketed with ambiguous labels, such as Natural Saffron, Selecto Saffron, Saffron Artisan, Superior Saffron, Product of ... (a certain EU country), etc. It is very likely that the use of stable isotopes as biomarkers of traceability is the technological solution in the future, to prevent fraud of origin of saffron and many other related products of PDOs and PGIs. The international consortium that support this COST action do not perform isotopes techniques, but -omics applied research aims to develop new insights into the DNA of saffron and other plant dyes, which will be useful for the detection of adulterants and strengthening the fight against fraud in Saffron industry.
Scientific work plan - methods and means
Based on these challenges, an intense coordination is required in order to approach the major fields on Saffron OMICS, to achieve synergy amongst experts, to avoid repetition or competition between groups working on the same area, and to contribute to the development of a RTD structure on this field. The Action will focus on main objectives, all involving advanced research tools and concepts:
-Genetics & Genomics, To investigate the degree of genetic diversity that exists within Saffron local varieties, those have undergone solely clonal reproduction. Then, genetic variation between Saffron clones will be analysed and the polymorphisms found further characterized to understand the nature and frequency of this genetic change that occurred during long-term vegetative propagation. We aim to clarify the genetic relationships among geographic or genetic groups both cultivated and wild (heterozygosity of varieties; identification of genetic bottlenecks, origin of C. sativus).
-Molecular markers assisting breeding, traceability and adulteration control. The goal is to provide molecular markers for the characterisation of Crocus germplasm, their application in selection of improved cultivars, and the development of analytical techniques as tools to support traceability in the different PDOs (La Mancha, Kozani, L’Aquila, Sardinia, San Gimignano, Firenze, & Mund), as well as specific markers to detect the presence of Gardenia and Buddleia adulterants.
-Transcriptomics & Metabolomics. The characterisation of the transcriptome of Saffron stigmas is the most vital to shed light on the molecular basis of flavour, colour biogenesis, genomic organization and biology of gynoecium of Saffron. The information derived can be utilized for constructing biological pathways involved in the biosynthesis of principal components of Saffron i.e., crocin, crocetin, picrocrocin and safranal.
-Phytochemistry. The development of chromatographic techniques (HPLC, LC-MS-MS, LC-DAD & GC), tristimolous colourimetry, infrared spectroscopy (FT IR, FT-NIRS), amongst others, and their introduction in the ISO/TS 3632-1 (2003) norm that regulates the purity and quality of Saffron, will improve the certification of authenticity of the spice, and the detection of adulterants.
Four work areas are proposed, differentiated on the basis on the use of different techniques and scientific
1. Nucleic acids work (genetics, genomics and transcriptomics).
2. Chemical work (phytochemistry, biochemistry and instrumental analysis)
3. Integration work (molecular and phytochemical markers for fingerprinting useful in breeding, traceability
4. Office work (dissemination, management, administration, coordination, etc.).