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 1st 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 above-mentioned 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 non-conventional 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.