Crocus, saffron-omics and the highest value crop

Saffron, the stigma of Crocus sativus, is the highest priced agricultural product (often €/$25 or £15 per gram) and a good example of a profitable crop with sustainability, cultural and social values, and high labour demand. I have been discussing –omics studies of the crop – the DNA, RNA, metabolites and secondary products – at the annual meeting of a European Science Foundation COST programme Saffronomics.

The ‘Action’ aims to coordinate research on Saffron-omics for crop improvement, traceability of the product, determination of authenticity, adulteration and origin to provide new insights that will lead a sound Saffron Bio-Economy. Despite the high price, the spice costs only a few pence/cents per portion, and adds enormously to the flavour and colour of many dishes. Biologically, saffron is the species Crocus sativus, as recognized by Linnaeus, and it is a sterile triploid with 2n=3x=24 chromosomes.

The programme of our Annual Meeting opened with the genomics sessions – the DNA, RNA, genetics and epigenetics. I don’t usually start reviews with, nor indeed include, my own talk, but here its content sets the scene for other work discussed at the meeting. I talked about the work of Nauf Alsayid, who shows the lack of any clear DNA differences between any accessions of saffron – whether from Kashmir, Greece, Italy, Spain, Holland or Iran. I cited a paper from 1900, itself reporting work back to 1844, where the French botanist Monsieur Paul Chappellier reported “for the Saffron, there is only known a single and unique species; for ages it has not produced a single variety”, writing that he was importing bulbs Naples, Athens, Austria, Spain, Cashmere and China (Chappellier P 1900. Creation of an improved variety of Crocus sativus. J. Royal Horticultural Society XXIV Hybrid Conference Report 275-277 – brilliant download, even available free for Kindle!). Plus ça change, plus c’est la même chose!

After my talk, Jean Marie Thiercelin, the seventh generation of the major saffron and spice company http://www.thiercelin1809.com told me that his grandfather knew Paul Chappellier, and he commented in the history of saffron production in France: Chappellier knew how to produce 10 to 15kg per ha before the First World War. After the war, saffron production stopped altogether in France, but it has restarted this century, with now some 137 growers on 37 ha but production of only some 5kg per ha.

Continuing with the talks, a DNA-sequence level study of saffron by Gerhardt Menzel with Thomas Schmidt (Dresden) analysed of several Gigabases of genomic survey sequence data, revealing about ten distinct tandemly repeated satellite DNA sequences that could be used to identify chromosomes in saffron by in situ hybridization. The species has a 78% repeat content in the DNA, with about 6% being the rDNA, and many different classes of transposons.

Giovanni Giliano (with Sarah Frusciante, Italy) demonstrated the carotenoid cleavage dioxygenase from saffron stigmas catlayses the first step in saffron crocin biosynthesis, a clear example of the pathway to the critical secondary product giving saffron its value (http://www.pnas.org/content/111/33/12246.short).

Both Matteo Busconi and Silvia Fluch (Austria) discussed epigenetic differences detected from different saffron collections: important for both understanding the controls on gene expression and for determining the origin of samples. Each producing area seems to have distinct profiles. Caterina Villa (Porto) reported results from use of the plant ‘barcoding’ primers ITS and matK with high resolution DNA melting analysis for saffron authentication, and more detail about the chloroplast genomes was presented from Bahattin Tanyolac and his Turkish colleagues. Although wild species of crocus are of interest from several points of view, only one paper, from Joze Bavcon (Slovenia) discussed these in detail, with a report of the natural hybrid Crocus reticulatus x C. vernus.

The next group of talks discussed the saffron metabolome, the analysis of different constituents of Crocus. Crocus is one of the few species to have its own international standard (ISO3632: http://j.mp/isosaffron ), and both quality and purity are measured (including contamination with stamens and pollen, along with detection of adulteration. Several participants were involved in the formulation of the standard, and Gianluca Paredi reported improvements that need less than the ISO methods needing no less than 23g of stigmas! Natural colours from plants such as Buddliea, Calendula, Curcum, Gardenia, safflower (Carthamus Asteraceae), cochineal (from the insect) and turmeric are widely mixed with saffron.

The Saffronomics project leader, Maria Tsimidou (Greece), used the three ISO3632 peaks for saffron – colouring strength from crocins absorbing at a peak wavelength of 440nm, aroma from safranal at 330nm, and taste (flavour) from picrocrocin at 257 nm – for examination of quality and authenticity of commercial saffron samples. Of 16 samples, 3 were adulterated, and half of the pure samples were graded in ‘category I’. Another amazing figure quoted was the price of saffron in quantity: of 75 tonnes imported to one county, only 35% is priced at more than $500 per kg. Authentic saffron could not be produced for anywhere approaching $1000/kg (typically $10-$15000/kg), so all this bulk product is fraudulent. Technology sessions in the meeting covered alternative quantification approaches to spectroscopy: Laura Ruth Cagliani in Milan tested  different solvents for extraction for NMR-based metabolomic characterization of authentic saffron distributed within the COST partners as well as the NMR evidence of absence of plant adulteration in those saffron samples.

A leading group from Thessaloniki was able to detect adulteration with as little as 15% cochineal. EA Petrakis and Moschos Polissiou demonstrated how FT-IR spectroscopy is promising to quantify small amounts of adulterants in saffron – safflower, Gardenia and tumeric – where diffuse reflectance mode provides rapidity, ease of use and minimal sample preparation. Other important reports discussed aging effects on profile of secondary metabolites (Paraskevi Karastamati Greece) and detection of herbicide residues (Christina Mitsi).

Micha Horacek (Austria) presented new results looking at the ratios of stable isotopes in saffron, a technique increasingly used to determine the origin of all agricultural produce. He showed the impressive map of with the gradient of water (hydrogen and oxygen) isotope ratio from North to South and from East to West in Europe. He also showed the differences in nitrogen stable isotope ratios depending of fertilizer use, and sulphur which depends on the underlying geology. Current work with saffron shows considerable year-to-year variation in the position of accessions from different regions of Europe, but the data is still being collected. Soon Micha will be getting a sample of our own, Leicester-lab-produced, saffron to add to his map!

Our hosts at RIKILT, the Food Safety and Quality Institute, Wageningen University, have much advanced applied science on food quality. An eye-opening talk by John van Duynhoven told us about rehydration of freeze dried blanched carrot with dynamic assessment of water movement in samples with and without blanching, freeze drying at -28 and -150C. Another series of images showed water transport and the impact of pre-cooking of rice, using magnetic resonance imaging MRI as a functional measurement of rice cooking. The final section discussed why crackers don’t crack: vapour transport during shelf life of crackers! Modelling of the nature of water transport links processing & formulation to the structure and on to functional and storage implications.

For ESF – COST projects, dissemination and public understanding are important, and participants were treated to a preview of a series of six school books about Fran Azafran and Franny Azafran by Manuel Delgado from Cuenca, Spain. I look forward to seeing these in full, and hopefully to their availability in other languages too.

Like the best of the projects, I feel that saffron science has moved in the last decade, (including research in the consortia www.crocusbank.org and www.saffronomics.org) with notable fundamental, technical and applied outcomes of our research. We know about its relatives and genome structure, key genes, metabolic processes and the key secondary products, and even understand epigenetic control, corm growth and dormancy. After 4000 years of being sold fake saffron, the fraudsters know now that we can test for saffron purity and quality!