Genetic Resources of Saffron and Allies (Crocus spp.)

Genetic Resources of Saffron and Allies (Crocus spp.) J.A. Fernández Laboratorio de Biotechnología, Instituto de Desarrollo Regional Universidad de Castilla-La Mancha Campus Universitario s/n Albacete E-02071 Spain Keywords: bank of germplasm, biodiversity, Crocus sativus, CROCUSBANK Abstract Saffron is the highest valuable food product and one of the oldest crops and medicinal plants. Saffron crop is subject to strong genetic erosion. Until now no international institution has taken the responsibility of the creation and maintenance of a collection representative of the genetic variability of the crop and its allies. Recently, the European Commission has approved an Agri Gen Res 2005 project on Genetic Resources of Saffron and Allies (Crocus spp.): CROCUSBANK, coordinated by this author. The objective of our consortium, composed by 14 groups of 9 EU and non-eu countries, is to create, characterise and exploit a germplasm collection (bank) in Crocus species, including Saffron crocus. This collection is an urgent need for Saffron breeders and farmers, as pointed out in the 1 st ISSBB (Albacete, Spain). The present paper explains the background, objectives, organization and technologies to be applied in the development of the project. IN MEMORIAN Dr. Fikrat I. Abdullaev (1943-2006). A brave, enthusiastic, charismatic and sensitive man; an original and wise scientist. Also a friend. INTRODUCTION Saffron is a Precious and Intriguing Species Crocus species are members of the family Iridaceae. The plants in this family are herbs with rhizomes, corms or bulbs. The family Iridaceae embraces about 60 genera and 1,500 species. The genus Crocus includes native species from Europe, North Africa and temperate Asia, and is especially well represented in arid countries of south-eastern Europe and Western and Central Asia. Among the 85 species belonging to the genus Crocus, C. sativus L. (Saffron) is the most fascinating and intriguing species (see Fernández, 2004). This is not only because it produces the well known Saffron spice, but for the numerous mysteries surrounding its origins. Questions as when it originated; the native area or areas; the ancestor species and the mechanisms of origin; the wild or naturalized plants; the infertility and consequent absence of fruit and seeds; remain to be explained. Greece (Crete) has been mentioned as probable origin of this old crop with probably more than 4,500 years of age. If its domestication occurred at more sites simultaneously or at different times is still not resolved. This is because Saffron is not known to be wild or spontaneous and can only be propagated by human help. In addition to being the most precious spice in the world, Saffron possesses a set of somewhat unique agronomic and eco-physiological characteristics including a relatively low water use, growth and development during fall and winter, a very low

harvest index, a generative phase which is followed by the vegetative phase of growth, and an economic yield which is produced prior to a significant vegetative growth. Furthermore it has three exceptional quality attributes, i.e. aroma, flavour and yellow dye, a set of characteristics bringing about its uniqueness for pharmaceutical, food and textile industries. Between 100,000 to 200,000 flowers are required to yield one kilogram of Saffron spice (about 900,000 dried stigmas). Medium yield are around 10 kg of Saffron/hectare, but varying very much between countries, lands, seasons, and agronomic practices. Saffron s high price is due to the much direct labour required for its cultivation, harvesting and handling. This fact has made progressively uncompetitive this crop in the more developed countries, and undoubtedly such tendency will extent to the currently producing leaders. Therefore, there is need for increasing Saffron production and quality to cope with an increasing demand and market differentiation. This will be achieved biologically by means of plants with more flowers per plant, flowers with a higher number of stigmas, increasing stigmas size or stigmas with an increased amount of dye and aroma. In a further step, a new approach could take place, the consideration of C. sativus as a source of phytochemicals and biopharmaceuticals. C. sativus is an autumnal flowering geophyte with corms that are covered by a tunic, dormant during summer, sprouting in autumn, and producing 1-4 flowers in a cataphyll with linear leaves. The flower has an underground ovary, a style 9-10 cm long, dividing at the top in three red trumpet-like stigmas (2.5 cm long) that once dried form the commercial spice Saffron. Flowering spans from late autumn until December according to climatic conditions. Cytological studies have indicate that Saffron is a triploid species which genome shows 3n=24, x=8 chromosomes. Its triploid condition allows vegetative multiplication, but not regular sexual reproduction. This is because meiosis and gamete development in triploids are irregular, resulting into many anomalies in sporogenesis and gametophyte development. Saffron is an Amphiploid with Probable Low Genetic Diversity One important aspect of our issue is to ascertain the genetic diversity of C. sativus. Saffron usually multiplies year by year by means of corms. Because corm multiplication does not induce genome variations with the exception of some mutation that in a triploid Saffron population are not easily detectable, all Saffron should be similar one to the other. Some authors believed that Saffron was once naturalized in small areas from where it was lost as a consequence of a change in land use. C. sativus was generally assumed to be of autotriploid or hybrid origin. Now we have several data that support the alloploidy of C. sativus being the parents C. cartwrightianus and C. hadriaticus, both with 2n=16 and present currently in Greece but not in overlapping areas. Other possible parents, e.g., C. thomasi, from Italy and Croatia, C. mathewii from Turkey, and C. pallasii ssp. haussknechtii from Iran-Iraq- Jordan, cannot be excluded. The complexity of the evolutionary history of the genus Crocus suggests an intensive species hybridisation and explosive speciation in Crocus evolution that could be on the basis of the origin of Saffron. We now are sure that Saffron is an allopolyploid but the localization of the hybridisation event has not been ascertained so far. If the event took part several times could have generated different amphiploids and, in consequence, different Saffron lines. Saffron was introduced in Western Europe in different historical moments: In the Iberian Peninsula by the Arabs or even before (Romans); in Germany, Switzerland, France, Italy and Great Britain by the Crusaders, and towards the east it was extended by a variety of cultures through

Transcaucasia, China, India and eventually Japan. Should we expect to find genetic diversity then? We can look the chromosomes as a first approach. The karyotype of C. sativus has been studied by a number of authors, reporting that Saffron from different countries (Azerbaijan, Iran, Italy, Turkey, France and England) was always 2n=3x=24 lacking karyologycal differences. However in the past literature other karyotypes were described. Cytofluorimetric analysis on nuclear DNA, carried to detect genome size and base pairs composition, revealed no differences in DNA content and composition in Saffron corms cultivated in different countries (Italy, Israel, Spain, Holland). Some assays with molecular markers (RAPDs) have revealed limited genetic differences among Saffron samples from Italy, Iran, Greece and Spain. Nevertheless, analysis of phenotype revealed differences in aspect flower size, tepal shape and colour intensity with lobed tepal in plants from Israel and more intense colour of tepals in plants from Sardinia (Italy). Variants of Saffron with an increased number of stigmas, maintaining 2n=24 have been reported with a frequency of 1.2x10-6 of the rare type flowers. Such phenotypes are well known by farmers and breeders but unfortunately they are not stable. Morphological differences with flowers having higher number of style branches and stamens have been already described in Saffron cultivation at L Aquila (Italy). Phenotypic variants are also present in Kashmir Saffron (F. Nehvi, personal communication). Besides different commercial products are known that could suggest the existence of different Saffron ecotypes or commercial varieties, the actual genetic variability present in C. sativus at worldwide scale is currently unknown. Nevertheless, there is a suspicion in Saffron breeders regarding the existence of scarce genetic variability in this crop, but no serious effort has been carried out to ascertain this important issue. They have been attempts with the objective to increase the spectrum of variability for floral traits and recovering auto-hexaploids (6x=48) in Saffron (to break the sterility barrier) by colchinization and to induce the genetic variability in Saffron using physical irradiation in order to develop polyploid forms; tetrafid, pentafid or hexafid stigmatic plants; and colour mutants. These attempts have been unsuccessful so far (for review see Fernández, 2004). THE RELEVANCE OF SAFFRON CROP Saffron is Highly Valuable Medicinal and aromatic plants have been increasing in importance to society continuously for the past 100 years. Saffron is made from the dried stigmas of the Saffron flower, a triploid sterile plant that is vegetatively propagated by means of bulbs (or corms). Saffron is mostly used as spice and food colorant and, less extensively, as a textile dye or perfume. However, due to its analgesic and sedative properties folk herbal medicines have used Saffron for the treatment of numerous illnesses for centuries. Saffron is considered to be the highest priced spice in the world (on average, 500 $ every Saffron kg). Its high value makes Saffron the object of frequent adulteration and fraud (see Fernandez and Abdullaev, 2004). Saffron is a Crop in Danger of Extinction in Many Countries Saffron is currently being cultivated more or less intensely in Iran, India, Greece, Morocco, Spain, Italy, Turkey, France, Switzerland, Israel, Pakistan, Azerbaijan, China, Egypt, United Arab Emirates, Japan and recently in Australia (Tasmania), Afghanistan and even Iraq. While the world s Saffron production is estimated in 205 tons per year, Iran is said to produce 80 percent of this total, i.e. 160

tons. Khorasan province alone accounts for 46,000 hectares and 137 t of the abovementioned totals, respectively. The Kashmir region in India produces between 8 to 10 t mostly dedicated to India s self-consumption. Greek production (4 t) is located exclusively in Macedonia (Kozani) and controlled by a single cooperative. Morocco produces between 0.8 and 1 t (Table 1). These figures do not pretend to be accurate (an impossible task for several reasons). They just want to be good enough to illustrate the reader. Saffron production has decreased rapidly in many European countries. Spain, the traditionally world leader and most reputed Saffron producer for centuries, nowadays makes about 0.3/0.5 t. Productions of Italy (Sardinia, Aquila, Cascia) 100 kg; Turkey (Davutobasi, Saffranbulli) 10 kg; France (Gâtinais, Quercy) 4/5 kg and Switzerland (Mund) 1 kg are nearly nominal. Other countries as Azerbaijan produce negligible amounts of Saffron. An illustrating fact: In early 1970s Saffron cultivation in Spain and Iran were 6,000 and 3,000 ha, respectively, while at present the surface areas are 77 ha in Spain and near 50,000 ha in Iran. Only 20 years ago Spain and Iran were producing the same quantity, about 35 to 40 t. Saffron crop disappeared in other European countries such as Germany, Austria and England; here it was grown in great quantities in Essex (especially near a town called Saffron Walden) and Cambridgeshire. After having been the leaders of Saffron production at commercialisation at a worldwide scale for centuries, nowadays European countries only produce a scarce 3 %, even tough the quality and the prestige in the marker still correspond to the European brands. The European Union has awarded the designation "appellation of origin" to the "Azafrán de la Mancha", the Greek "red Saffron" under the name "Krokos Kozanis", and the Italian "Zafferano dell'aquila". The reasons of Saffron decadency are various. An intensive (and expensive) hand labour of up to 15 working days per kilogram of dry Saffron spice is required for flower picking and stigma separation. To the high cost of this labour it should be added the very uncomfortable stooping position of the flower pickers, and the very short picking period which comprises the early morning hours of the 20-30 days of duration of the flowering season. The mechanisation of flower picking in field grown Saffron has proved difficult. All Saffron producers in the EU, also soon Turkey, suffer from increasing labour costs. Iran can increase its production to more than 200 t whereas India could offer its Kashmir Saffron to the world market in growing amounts. China will become a massive maker and there are serious projects of Saffron production in Afghanistan and Iraq. A grey market of Saffron has developed in some countries in the Caucasus, trading Iranian Saffron through doubtful channels without quality control. Countries in North Africa are the primary origin of forged Saffron, mostly Carthamus tinctorius or Curcuma. Hence, the Saffron world market panorama is al least uncertain. Nevertheless, although the tendency of Saffron diminution has been constant, they are symptoms of a revival in Saffron crop in Europe. France, for instance, has shown the emergence of new associations of Saffron farmers ("Les safraniers du Gâtinais" in 1987, and Les Safraniers du Quercy in 1999) after decades or even centuries of abandon of the crop. Other initiatives are flowering in Italy (Sicily) and in many other countries outside Eurasia such as Australia (Tasmania), New Zeeland, Argentina, Chile, Bolivia, even USA (Pennsylvania). Other Crocuses are also Economically Important The Crocus genus is known mainly for the cultivated species C. sativus, which is of prime economic importance. However, there are also other species belonging to

this genus, which are highly prized for their colourful flowers, and thus used extensively in specialized gardening (Table 2). These are horticultural varieties of C. vernus, C. versicolor and C. aureus, amongst others. Most of the Crocus species grow naturally in the fields between shrubs and grass or light woodlands. The plants in this family are herbs with rhizomes, corms or bulbs. The genus Crocus includes about 80 species distributed from south-western Europe, through central Europe to Turkey and south-western parts of Asia, as far east as western China (Mathew, 1982). Saffron is Subject to Strong Genetic Erosion As explained, the lost of land surface dedicated to Saffron crop in many areas has resulted in a corresponding genetic erosion, the situation being dramatic at the present time. Traditional plant breeding techniques are based on a bulk selection of the best samples among natural or cultivated populations; genetic breeding with wild ancestral species; and spontaneous or induced mutations. Sterility in Saffron limits the application of conventional breeding approaches for its further improvement. Besides different commercial products are known that could suggest the existence of different Saffron ecotypes or commercial varieties, the actual genetic variability present in C. sativus at worldwide scale is currently unknown (Fernandez, 2004). They have been efforts by Indian researchers to increase genetic variability in Saffron using nonconventional breeding techniques, such as induced mutagenesis employing physical irradiation and induction of polyploidy by colchinisation. Nevertheless, the preliminary results of induced genetic variability are not completely hopeful and probably would require further work. A CROP OF ADDED-VALUE FOR EUROPE Saffron must be considered as an endangered crop of added value deserving scientific interest in Europe for various reasons. Its Origin and Historical Background The origin of Saffron crop is uncertain. C. sativus is cultivated for its spice for at least 3,500 years in Egypt and Middle East. The name Crocus finds its origins in the Greek word krokos, Saffron, which in turn derives from the Semitic word karkom, one of the oldest names for this plant. Some archaeological and historical studies indicate that domestication of Saffron dates back to 2,000 1500 years BC. This is derived from documents reproducing the plant or showing people collecting the crop. However the sites where the first Saffron plants appeared differ according to the opinion of various authors. Vavilov placed Saffron into IV plant origin centre of Middle East (Minor Asia, Transcaucasia, and Turkestan); whereas more recent contributions indicate that the process of Saffron domestication has to be identified on Crete during the Late Bronze Age. Historical records detail the use of Saffron date to ancient Egypt, Persia, Greece and Rome, where it was used as a medicinal plant, as a dye, in perfume, and as a spice and colorant for culinary purpose. Saffron use reached its highest point in the Iberian Peninsula by the Arabs. During the Middle Age Saffron crop was extended for rest of Europe due to the Crusaders, and was settled in Switzerland, France and Italy, reaching Great Britain in the 14 th century. Towards the east, Saffron crop was preserved for diverse civilizations in the current territories of Iran, Azerbaijan and Kashmir. Saffron was firstly exported to China in 13 th century as healthy food, and again in 16 th century as medicine. It reached Japan at the beginning of 17 th century. Since then, Japanese people have used Saffron almost exclusively as health product.

Tradition and Quality Besides the European countries only produce a scarce 3 % of world Saffron market, the quality and the prestige in the marker still correspond mostly to the European brands. La Mancha Saffron has been generally the highest prized, and La Mancha region was during centuries the world biggest producer. Currently the Spanish manufacturers and dealers control 70 % of the world s Saffron market. The European Saffron under Protected Geographical Indication (PGI) status are: "Krokos Kozanis"(EC Reg. 378/1999), Greece (Kozani) Azafrán de la Mancha" (EC Reg. 464/2001), Spain (La Mancha) "Zafferano dell'aquila" (EC Reg. 2081/92), Italy (L Aquila) Munder Safran AOC, Mund (Switzerland) France have two associations of Saffron farmers that pursue the same PGI status: "Les safraniers du Gâtinais" in 1987 Les Safraniers du Quercy in 1999 As mentioned Saffron crop is in severe danger of extinction in Europe at the present time. What used to be the King of Spices suffers from a worldwide devaluation. Amazingly, very little has been done to protect the Saffron crop in Europe. Recently, in 2002, the Regional Government of Castilla-La Mancha (Spain) spent 150,000 euros/year in direct financial support to Saffron producers, ranging between 1,200-1,800 euros/ha with a maximum of 3,000 euros per farmer. Apparently, such benefit has not continued in the subsequent years. Anyhow, the genetic erosion has been intense and now it now it is urgent to protect what remains of the European Saffron germplasm. On the other hand, Saffron is considerate to be the highest priced spice in the world. In fact, Saffron is one of the most valuable food products (the red gold), and its highest quality is still associated to the European brands. Although not exclusively, the European production has the added value of prestige and cultural significance of the product (gastronomy, history, art). Food Safety Consumers are confused with the differences in quality between Saffron of different origins and the subsequent fluctuation of prices. The quality of Saffron is certified in the international trade market following the ISO 3632 Normative since 1993. The most important parameter is colouring strength, calculated from UV-Vis measurements at 440 nm in aqueous extracts of this spice. Such measurements are related to the total carotenoid content. This regulation is currently under controversy since leave aside the most important organoleptic properties of Saffron (odour, flavour) and does not prevent fraud. Saffron commands a rather high value in the international spice trade that results in its frequent adulteration by artificial colorants and by mixing genuine stigmas of Saffron flower with other parts of plants (e.g. some species of grass) artificially coloured. Molecular tools will be ideal for checking purity of product, even after processing, act as markers for adulteration (either with dyes or with other plant species). Alternative Uses Saffron is mostly used as spice and food colorant and, less extensively, as a textile dye or perfume. However, due to its analgesic and sedative properties,

traditional Eurasian herbal medicines have used Saffron for the treatment of numerous diseases. Saffron has been indeed one of the oldest plants used as medicine (see the frescoes of Thera, Greece). The renewed importance of Saffron as nutraceutical is being sustained by an increasing number of evidences. Saffron has been claimed to have effects on senile dementia, retina-degeneration, immunomodulation, as well as antimicrobial, antidepressant, antitumour or cardiovascular protective properties. Although these effects required more pharmacological/nutraceutical trials and significantly additional scientific substation, we support the potential health application of this plant, with a profile similar to green-tea, ginseng, etc. The application of the agroresource refining concept to the other parts of the Saffron plant (style, petals, stamens, leaves, stem and corms) is actually underway in order to valorise all the organs of the plant. This Saffron by-product valorisation is in agreement to the European Agricultural Policy that tries to encourage alternative uses for agricultural matters. Some of our research groups are focused in obtaining pharmaceutical, aromatic and dye products for health and cosmetic industries (Fernández and Abdullaev, 2004). Sustainability Saffron is a low water demanding plant well adapted to arid or semiarid lands; it has a low input of fertilizers (if any) and chemicals. Actually, Saffron is still cultivated almost in the same manner for 3,000 years. This environmentally sound crop is one of the ancients of Europe. Its unique eco-physiology amongst many crops makes this plant appropriate for marginal soils, mainly but not exclusively in the Mediterranean area. The concept of a sustainable EU agriculture fits perfectly with the Saffron crop. WHAT TO DO TO PREVENT SAFFRON DECLINE The majority of the germplasm collections include crops with a high economic value like cereals, legumes, fruits and forage species. However, is rare the presence of species with neutraceutical, therapeutic or medical applications, aromatic plants, ornamental and spice producing plants in public collections. The limited genetic variation suspected for this sterile crop, exclusively propagated vegetatively and subject of rapid genetic erosion in the last century, would have required the creation of a collection of landraces, ecotypes or simply accessions of C. sativus. However, no germplasm collection of Saffron in Europe neither in the world has been created so far. The creation of such collection will contribute not only to slow down the intense genetic erosion but also will make available a wide variety of Crocus genotypes of potential carriers of interesting genes for plant breeders, e.g. resistance to biotic or abiotic stresses, reserve accumulation, biosynthesis of secondary metabolites, etc. In order to ensure the future of Saffron crop it is necessary to improve cultivation techniques, plant material, quality evaluation methods, and to develop a wide range of Saffron uses particularly those related to human well-being. The worldwide increase in utilization of Saffron as natural product requires new biological and economical development, and co-operative programs on technological and medicinal studies. Production and processing of medicinal and aromatic plants should be one of the successful branches of horticulture in the future. To fulfil these requirements, however, production systems need to be modernised and high quality material must be provided to farmers. The creation of a Bank of Germplasm in crocuses could help in that purpose

The 1 st International Symposium on Saffron Biology and Biotechnology held in Albacete (Spain) and organised under the auspices of the International Society For Horticultural Science and the University of Castilla-La Mancha, counted with the participation of one hundred researchers, technologists and businessmen from 15 countries (Spain, Iran, Mexico, India, Greece, Italy, France, Japan, Switzerland, USA, Turkey, Hungary, Denmark, Canada, and Azerbaijan). The meeting, chaired by this author provided a worldwide overview of research in Saffron achieved in the last decades, including basic biology, agronomy, genetics and breeding, chemistry, industrial production, pharmacognosy, pharmacology, and economics. Attention was also given to the scientific and practical problems and the challenges for Saffron plant in the XXI century. One of the statements of the Working Group on Saffron Biology and Biotechnology (M04), created at this meeting inside the Section of Medicinal and Aromatic Plants of the ISHS, was to promote the creation of a Bank of Germplasm and Gene Banks in Saffron to preserve genetic biodiversity in Saffron and related species (Fernández and Abdulalev, 2004). A World-Scale Task Note that while the world s total annual Saffron production is estimated at about 200 tons per year, Iran with more than 47,000 hectares of land under Saffron cultivation is said to produce more then 80 percent of this total. India is the second producer. Any serious effort to create a bank germplasm in Saffron would be enriched with the participation of researchers, agronomists or merchants from these countries, simply because they must keep a significant amount of the global genetic variability present in Saffron crop, together with a wide knowledge of its agronomy and uses. It must be strongly taken into account that having the support of research groups from countries that are putative commercially competitsors is highly valuable. This confidence is based on the environment of collaboration and frankness amongst scientists generated in the 1 st ISSBB. A new big step has been done with the 2 nd ISSBT carried out at the Ferdowsi University of Mashhad (Iran) in October 2006 which contents are included in the current Acta Horticulturae volume. It is not an overstatement to say that this Bank of Germplasm extents its obvious European interest to a wider scale, and must be the basis of the World Saffron & Crocus Collection for everybody s use. A FIRST MILESTONE: THE CROCUSBANK PROJECT Agricultural Policies Environmental concerns play a vital role in the agricultural policy of the European Union (EU). By managing a large part of the European Union's territory, agriculture preserves many specific genes, species and habitats. The EU set up measures in support of the agricultural biodiversity, to contribute reaching the 2010 target of halting the loss of biodiversity. In the Biodiversity Action Plan for Agriculture (Council Regulation (EC) No 870/2004) it was proposed to launch a new EU programme on the conservation, characterisation, collection and utilisation of genetic resources in agriculture (2004-2006). This programme promotes genetic diversity and the exchange of information including close co-ordination between Member States and between the Member States and the European Commission for the conservation and sustainable use of genetic resources in agriculture. It also facilitates co-ordination in the field of international undertakings on genetic resources in agriculture, in particular within the Convention on Biological Diversity (www.biodiv.org/default.shtml) the International

Treaty on Plant Genetic Resources for Food and Agriculture (www.fao.org/ag/cgrfa/ itpgr.htm) and the FAO's Global Plan of Action for the Conservation and Sustainable Utilisation of Plant Genetic Resources for Food and Agriculture (www.fao.org/waicent/search/5_dett_fao.asp? cgiar=&calling=simple_s_result&publication=1&webpage=2&photo=3&press=5&vi deo=9&lang=en&pub_id=57930). The principle of The conservation and sustainable use of genetic resources in agriculture are essential to the sustainable development of agricultural production and of rural areas prompt the Saffron germplasm main stakeholders in the European regions and third states around the world to joint a consortium involving crocus relatives to propose the so-called CROCUSBANK project on GENETIC RESOURCES OF SAFFRON AND ALLIES (CROCUS SPP), aiming to contribute to the Biodiversity Action Plan for Agriculture. The CROCUSBANK project will play a part to the new Programme on the Conservation, Characterisation, Collection and Utilisation of Genetic Resources in Agriculture. AGRI GEN RES launched by the European Commission in 2005. The recent approval of this 4-years length project constitutes a big success for all Saffron researchers taking into account the strong competition of several projects dealing with animal and plant species. The objective of this project is to create, characterise and exploit a germplasm collection (bank) in Crocus species, including Saffron crocus. These are the goals. 1. Collection, multiplication, conservation and documentation of Crocus genetic resources. This collection has two main goals: First, to collect and reproduce Saffron bulbs, coming from all the countries that cultivate Saffron, for direct use of this plant material in selection programmes all over the world; and second, to create a collection of Saffron allies for conservation, since they are endangered and threatened taxa and populations in Crocus, and for research in taxonomy and evolution, genetics, physiology, ecology and agronomy. The Crocus species are exploitable sources of resistances and other agronomical interesting traits to be transferred to Saffron, through appropriate breeding programmes and technological tools. Resistance to fungi and other stresses are actually priorities in Saffron breeding. The general objective can be divided into the following actions: Exploration and collection of germplasm of Saffron and related species. The collection of Crocus material will be carried out by means of requests to different regional centres growing the plants and visiting specific locations at appropriate date to collect both cultivated Saffron and wild species. The taxa included in this project shape a proposal of maximum achievements. The reality of the field work is taken into account regarding the number of Crocus species and subspecies that we are able to collect. Elaboration of a list of descriptors for the characterisation of the genus Crocus and primary characterisation of the collected material. Multiplication of the collected plant material for its conservation in the Bank of Plant Germplasm of Cuenca (Spain). Conservation methods based on tissue culture techniques will be used when required. Elaboration of an effective documentation system, with the passport and characterization data of the accessions, in order to guaranty an appropriate management of the Crocus germplasm collection. To make available this material to potential users by distribution of corms, tissue culture and DNA samples. 2. Characterization and evaluation of Crocus genetic resources. We will elaborate a list of descriptors for the characterization of the genus Crocus and primary

characterization of the collected material. For the characterization/evaluation of the material we will take into consideration phenotypic characters with good heritability at different structural and physiological levels and include both simple, single-gene autoapomorphic characters and complex quantitative traits: Morphological (floral features, corm size); Phenological (flowering and relationship of climate, latitude and altitude); Cytological (chromosome numbers, genome size, ploidy level and identification of hybrids); Phytochemical (Saffron chemical composition; metabolic profiling); Molecular (DNA analysis); and Physiological (abiotic stresses and pathogen responses). 3. Application of the Crocus germplasm information and banked accessions. Rationalization of collections, identifying duplicates in order to optimise the management of the bank. Definition of valuable germplasm for Saffron breeding. Identification of ecologically rare and important species/genotypes in the natural environment. Identification of valuable species, cultivars and hybrids for the horticultural industry. Comparative genomics with model and crop species to identify universal features and valuable genes for agronomy. The Consortium The CROCUSBANK consortium (Table 3) is composed by partners from 6 European Union countries and 3 partners from third countries where rich resources of Saffron and Crocus relatives are found. All of them have long experience in research on complementary aspects of the Saffron and/or Crocus biology, agronomy, and biotechnological applications. The consortium tries to joint a wide representation of the effort carry out currently on Saffron research at international level, and the partner s geographical distribution comprise the Saffron producing areas as well as the main habitats of the Crocus species. The non-presence of Iranian and Indian groups is explained in terms of administration inconveniences. The AGRI GEN RES programme does not subsidize groups from third-countries, which nevertheless are free to participate without funding, as partners from Azerbaijan, Egypt, and Turkey have done. Besides, having the collaboration of Iranian and Indian scientists and institutions is highly desirable and we look forward to accomplish it. All scientific, financial and administrative aspects of CROCUSBANK will be co-ordinated by this author at the University of Castilla-La Mancha (Spain), who will represent the consortium in the European Commission. The aim is to collect and reproduce Saffron bulbs, coming from the EC countries that still cultivate Saffron (Spain, Greece, Italy, France, and Hungary), but also from Turkey, Azerbaijan, Morocco, India and Iran. This plant material, once adapted and reproduced, could be used in selection programmes all over the world. Secondly, the collection of Saffron allies (both seeds and/or corms) will be made for conservation, since some Crocus taxa are threatened, and for research in genetics, plant breeding and basic biology. The creation of the mother collection in Spain will be followed by replicas in other countries in the coming years, once the bank is well established. Coordinated Implementation Plan The action comprises three basic tasks: collection, conservation, evaluation and utilisation of Crocus genetic diversity. The first duty will be completed by the participation of 12 groups of the consortium. Each group has the responsibility to

collect the quoted plant material using botanic gardens, personal contacts, other collections and explorations to the field. The Crocus wild species that are included in the list are the complete genus (Table 5). Explorations and collection per area are designed on the basis of the geographical proximity of both Saffron production areas and Crocus spp. population habitats. The programme of collection of Crocus taxa is planned on a basis of maximums, in order to obtain different subspecies and populations of each species. Partial results are not considered a failure since the purpose is to obtain as many accessions as possible, representative of the species range and diversity, according to the financial and human resources available. The conservation, multiplication, maintenance, documentation and management of the collection will be carried out by the JCCM Group, centre specialised in the management of germplasm banks of the Castilla-La Mancha region in Spain, amongst them, the dedicated to aromatic and medicinal plants. According with the objectives of the project, translated into workpakages, various overlapping groups are established. The first one, named Saffron Collectors Group (SCG) is integrated by partners 0, 4, 5, 6, 7, 8, 9, 11 and 12, under the supervision of partner 0 (UCLM, Spain). The second is the Crocus Collectors Group (CCG) composed by partners 0, 4, 5, 7, 8, 9, 10, 11, 12 and 13, under the command of partner 11 (NAGREF, Greece). The partners 1, 4, 5, and 10 constitute the Descriptors Group (DG) whose manager is partner 4 (UPVLC, Spain). The Multiplication and Conservation Group (MCG) is organized by partners 0, 1, and 10, with partner 1 (JCCM, Spain) as manager. The characterization and evaluation task is approached by two groups: (1) morphological, physiological and agronomical level, with partners 0, 1, 4, 5, 7, and 10, leaded by partner 1, and (2) chemical and molecular level, with partners 0, 2, 3, 6, 8, 9, 10, 11, 12 and 13, under the organization of partner 2 (AUA, Greece). These groups are called CEGA and CEGC, respectively. The coordination of the complete characterisation task will be made by the project coordinator (partner 0). The application of the germplasm information and banked accessions is implemented by the Application Group (AG) integrated by partners 0, 1, 3 and 10, leaded by the latest (ULEIST, UK).The management and coordination of the project) is the responsibility of the project coordinator (partner 0) assisted by the Project Management Committee, PMC (partners 1, 2, 4, 10 and 11). Partner 0 (UCLM, Spain) is the overall project coordinator, whereas partner 1 (JCCM, Spain) is the manager of the collections and the central information system. SCG activities consist in the collection of Saffron corms from different EU and non-eu countries, in an attempt to store the maximum genetic variability present in this crop. The wide commercial experience and good knowledge of Saffron cropping areas of the French company (TJMT) (partner 6) will be extremely convenient for this achievement. CCG task is the collection of Crocus taxa by means of requests to different collection holders or visiting specific locations at appropriate date to collect either cultivated or wild specimens (seeds and/or bulbs). It is of particular interest to collect a wide representation of taxa phylogenetically close to Saffron (i.e. Crocus series of the genus) for their interest in Saffron breeding. These species are present in Greece and its islands. The Greek sub-coordinator (partner 3) has a long experience in C. sativus studies and incorporates experienced geneticists and plant breeders. Included in partner 10 we gain the participation of the Crocus Group (historically) a splinter group from the British Iris Society, that is an assemblage of persons with a shared interest in the genus Crocus. The group was created in 1974 and includes botanists, Crocus growers and people dedicated to

computing, seed exchange and other duties (www.thealpinehouse.fsnet.co.uk/crocus %20pages/). Anthony Goode is coordinator of the Crocus Group and will be subcontracted by partner 10. He is an expert in cropping conditions and techniques for a big number of Crocus. His valuable practical knowledge is of major interest for the Crocus germplasm project. The experience of partner 4 (DG coordinator) in Saffron experimental cultivation will be useful for the elaboration of descriptors and primary characterization of the plant material. The activities of MCG are the core of the project. Partner 1, its coordinator, has wide experience in the management and conservation of genetic resources in Spain. Lastly, CEGA and CEGC, coordinated by partner 1 (Spain) and partner 2 (Greece), respectively, must initiate the characterisation of the material at different levels, with the aim of continuing this task in the coming years. Partner 10 (UK) will be the manager of the application task (definition of valuable germplasm for Saffron breeding, identification of important accessions in ecology, horticulture, agronomy, industry, etc. Partner 11 (Greece) has a long experience in the management of genetic resources in this country. MAIN METHODOLOGIES Exploration and Recollection 1. Preparation and Execution of Expeditions. With the aim to obtain genetic material (corms) of cultivated Saffron, as well as seeds and occasionally corms of other Crocus species in their areas of distribution to be part of the germplasm bank, a previous work of planning of expeditions will be carried out. For cultivated species (some of them used in gardening) we will contact plant nurseries, botanic gardens and private cultivators. In the case of wild species we will make a complete bibliographic search on the different species in order to determine their chorology, dates and locations where the material could be found. Recollections will include natural habitats, crop lands or even local markets. The distribution of areas by groups will be tentatively as described in the next table, taken into account that changes will made on the way. The distribution of geographic areas, the taxa and the groups responsible of the exploration are explained in Table 3. The exploration of Saffron corms will start at early 2007, with the aim to carry out their sowing at the end of spring, and will be maintained through the years of extent of the project. Since wild species can only be detected in flowering time each group responsible of a geographic area should carry out four collecting campaigns of about two weeks per campaign during the autumns of 2007 and 2008, and the springs of 2008 and 2009, in order to collect Crocus species flowering in spring or autumn. 2. Elaboration of protocols for collecting material. The protocols of recollection of cultivated and wild species will be elaborated previously. These protocols must detail the information that must be compiled in the moment of recollection and associated to each germplasm sample: botanical data, ecological and geographical data, uses, crop techniques and any other observation of interest. They also will include the methods of collecting samples and identification of each of them. Conservation and Multiplication Most of the wild Crocus species are markedly entomophilous, fertile and seed producers. Nevertheless we will use the same method as for Saffron for conservation of accessions; i.e. the maintenance of corms in vegetative form, in order to guarantee the genetic stability of material, avoiding hybridisation between accessions.

The corms obtained in the campaigns of recollection will be multiplied in the field with the aim of produce a suitable minimum amount that guaranties their longterm conservation in the germplasm bank and the supply to potential users. For the case of accessions of wild Crocus with scarce material, this will be previously multiplied in acclimatised greenhouse with the substrate of the collecting zone, in order to make sure the propagation of some material under the best conditions before the transference to the field, to minimize the risk of loss. In some cases the conservation/propagation in the greenhouse will continue. Multiplication will be carried out in a fenced parcel in the experimental farm of the Bank of Plant Germplasm of Cuenca (partner 1), with a medium altitude between 950 an 1000 meters, medium temperature of 11.5 ºC, and a precipitation ranging from 550 to 600 mm mainly concentrated in spring and autumn. The edaphic characteristics in this parcel are typical of the area, sandy loams, alkaline ph (7.6-8.4), normal electric conductivity (<400 mmhos/cm) and a low content in organic matter (1-2.5 %). Conventional labouring will be used to prepare soil for seeding (disk harrow, cultivator and rotary harrow). Each accession will be seeded in a 12 cm furrow; with 20 cm between plants and 40 cm amongst furrows. Weeding out will be carried out when required. The collection will be kept in the field indefinitely but every two years the corms of half furrow will be collected and reseeded to obtain supplying material. The other half furrow will be treated in the same way the next campaign. In cases of Crocus accessions with low fertility, difficulties for conventional multiplication, or in cases of endangered and threatened taxa and populations we will develop methods for in vitro conservation. In vitro Multiplication To establish the in vitro conservation of the distinct genotypes of Saffron and some wild Crocus collected in the development of our project, we will apply the methodology reported by Piqueras et al. (1999). Briefly, healthy corms will be selected from the collection and will be used as source of tissues as starting material in two developmental stages: (i) vegetative dormancy previous to sprouting and (ii) vegetative growing; in order to evaluate the impact of the physiological stage of corms in response to in vitro morphogenesis. The Saffron micropropagation protocol includes the following steps: Initiation of the culture and explant selection, Multiplication, Multiplication of meristematic nodules in liquid medium (optional), Elongation and rooting, Acclimatization to ex vitro conditions and formation of corms, Control of genetic stability and clonal uniformity of the regenerated material. Elaboration of a List of Descriptors and Characterization of the Collection Due to the local and minority character of Saffron crop there is no list of descriptors published by the International Plant Genetic Resources Institute (IPGRI, http://www.ipgri.cgiar.org/index.htm). For that reason, one of the basic goals of this project will be the elaboration of such a list. We will try to construct a list of descriptors valid for the genus Crocus, based on the taxonomic criteria for the genus, but also including the categories present in other IPGRI list of descriptors. The descriptor resulting of this project must be as complete as possible. The primary characterisation assays (morphological and agronomical traits) will be basically carried out in the experimental farm of Bank of Plant Germplasm of Cuenca during three consecutive years and with an identical design to the multiplication parcel (12 cm furrows; 20 cm between plants and 40 cm amongst furrows). A portion of the

corms will be collected each year for characterisation. The size of the samples will depend on the variability observed for the characters evaluated. Corms (and/or seeds in the case of fertile crocuses) extracted from the field of characterisation, together with those of the field of conservation/multiplication, will serve as supply to the potential petitioners in this campaign. The parcel of characterisation will be laboured as that of multiplication, but both will be located on separated zones of the fence, and will serve as replicas of security for contingencies. Documentation All the information associated to the Crocus collection will be included in a relational database in order to guaranty its correct management and availability for the potential users. Such database will be designed attending to recommendations and rules internationally established for documentation in plant germplasm collections and will be accessible via Internet through the Website of the Servicio de Investigación y Tecnología Agraria de Castilla-La Mancha (Spain) (www.jccm.es/agricul/paginas/desarrollorural/investigacion/cisita.htm). The data to be included in the database will be divided in the following categories: 1. Passport data. Which will be referred to geographical and ecological indicators of the recollection site. They would have been recorded in the recollection instant. 2. Management data. That will include the information generated in the process of conservation, multiplication and supply. 3. Characterisation data. Including accession identification, place of characterisation, characterisation data s. str. e the nature of an active collection and in its management a registry of incomes and outcomes of plant material will be carried out. The collection will have the nature of an active collection and in its management a registry of incomes and outcomes of plant material will be carried out. Molecular and Cytological Characterization The objective is to provide molecular and molecular cytogenetics markers for the characterization of Crocus germplasm and application in selection of improved cultivars for Saffron production. Thus, the molecular characterization of Crocus will allow: To identify clearly the off types in some collections To appreciated the extend of diversity of various geographic or genetic groups cultivated and wild. To clarify the genetic relationships between these groups (heterozygosity of varieties). A range of appropriate molecular marker are available for such purposes: Microsatellites (SSRs), inter-ssr and inter-retroelement methods, and gene based markers (PCR and hybridization based) will be used. A wide range of accessions of both cultivated Saffron and wild species will be used for characterization, and one or two outgroups will be included in analysis (Romulea and Iris) to allow robust rooting of phylogenies. Standard primers for phylogenetic analysis will be used to amplify gene segments from nuclear, chloroplast and mitochondrial genes. Molecular-based trees will be constructed to infer relationships between groups and accessions. Molecular-

based analysis will show how much diversity is present in the genus; this data will be in a form that it can be used to develop conservation policy. In addition to estimating genetic diversity, these tools can be utilized to establish molecular markers for phenotypic traits of interest. Genes which are widely conserved between species, with a focus on those associated with biotic and abiotic stress tolerance, will be amplified and sequenced from a range of accessions, using conserved sets of PCR primers. Genes associated with corm development and dormancy will be isolated and characterized from a range of accessions. Gene expression analyses, will allow to found more efficient alleles. Genes associated to flavour and other associated quality traits important in Crocus commercialization. Several genes directly involved in flavour have been identified in C. sativus, and several biochemical markers are used to determine the quality of flavour and aroma in Saffron, we would like to correlated molecular and biochemical flavour traits. Phytochemical Characterization...Saffron major constituents are crocins, picrocrocin and safranal, which are responsible for its colour, taste and aroma, respectively. Quality control of Saffron is based on chemical analysis that should discriminate such different compounds. Except for the method currently recommended by the International Standardization Organization, namely UV-Vis Spectrophotometry, information on the chemical composition is derived by HPLC combined with spectroscopic detection means (diode array, MS). Chemical composition of the polar fraction extracted from the stigmas of wild Crocus species will be characterized by the means of UV-Vis and HPLC-UV techniques. Further investigation on the chemical composition of stigmas of species with particular commercial interest will be based on the examination of other extracts (petroleum ether, diethyl ether, acetone, methanol, water). A typical UV-Vis profile of C. sativus extracts will be constructed for each of the different extracts and will then be used as a reference one for the characterization of the wild species. UV-Vis spectra derivatives of the individual extracts are also expected to highlight any deviation from the typical profile of C. sativus. In all cases, the quality parameters suggested by ISO 3632-2 (2003) such as colouring strength, bitterness and safranal content will be also determined. Main target of this work is finally the identification of constituents that could be a discriminative key for the characterization of different Crocus species to appreciate the extend of diversity of various geographic or genetic groups cultivated and wild. The essential oils (EO) content of the Crocus stigmas will be determined and the chemical composition of the produced oil will be characterized by using chromatographic and spectroscopic techniques such as GC-FID, GC-MS FT-IR, FT- Raman and UV-Vis. The hydrodistillation and the microsteam distillation - extraction to organic solvent will be used as reference methods for the production of EO. The ultrasound based assisted extraction and the microwaves based distillation will be the innovator methods for the EO production. These methods are expected to limit the time demand for the oil isolation receiving similar quality oil with the conventional methods and in some cases to improve the quality of the produced oil, especially by receiving enriched in active components extracts. The influence of temperature, solvent, frequency and time to the EO production will be taken into account in order to obtain the highest EO yield. Experiments will be performed with different solvents in variable extractable times, using ultrasound or microwaves apparatus, which will be functioning at specific frequency. Extractions based on ultrasound will be