In Vitro Micropropagation of Saffron C. Karaoğlu, S. Çöcü, A. İpek, İ. Parmaksız Department of Biology Faculty of Science and Arts University of Gaziosmanpaşa Tokat Turkey S. Uranbey Central Research Institute for Field Crops Yenimahalle Ankara Turkey E.O. Sarıhan Department of Field Crops Faculty of Agriculture University of Mustafa Kemal Hatay Turkey N. Arslan, M.D. Kaya, C. Sancak, S. Özcan B. Gürbüz, S. Mirici, C. Er, K.M. Khawar Faculty of Education University of Akdeniz Antalya Turkey Keywords: Crocus sativus, surface sterilization Abstract Saffron has been produced and exported from Turkey until nineteenth century. However, at the present time it is produced only in a few villages. Since saffron is sterile triploid, it is propagated by corms as the propagation through seed is impossible due to non setting of seeds. The natural propagation rate of most geophytes including saffron is relatively low. In vitro techniques have been used for the micropropagation of various plant species. In the present study various explants including floral and corm segments were cultured on different nutrient media supplemented with various concentrations of plant growth regulators. New corms were produced on MS medium supplemented with 2 mg l-1 6-benzylaminopurine (BAP) and 0.5 mg l-1 -naphthalene acetic acid (NAA) after 6 months of culture. Regenerated corms were kept at 5 ºC for 5 weeks and then transplanted to a potting mixture. INTRODUCTION Saffron is an important spice derived from the stigmas of Crocus sativus, a species belonging to the family Iridaceae. The domesticated saffron (C. sativus), a fall-flowering perennial plant is a sterile triploid mutant of the eastern Mediterranean fall-flowering Crocus cartwrightianus.(Deo, 2003) The stigmas are dried for use in medicine, food seasoning and coloring since centuries and are characterized as the most expensive spice by weight. Saffron contains more than 150 volatile and aroma-yielding compounds. It also has many nonvolatile active components, many of which are carotenoids, including zeaxanthin, lycopene, and various α- and β-carotenes. However, saffron's golden yellow-orange colour is primarily the result of α-crocin (Abdullaev, 2002). Saffron has been produced and exported from Turkey until nineteenth century. However, due to number of socio economic and technical problems, the cultivation of saffron has been limited to a few villages in the Safranbolu province of Turkey. Saffron is propagated by corms as the flowers are sterile and fail to produce viable seeds. A corm survives for only one season, producing up to ten "cormlets" that eventually give rise to new plants (Deo, 2003). Therefore, reproduction is human dependent; the corms must be manually dug up, broken apart, and replanted. The natural propagation rate of most geophytes including saffron is relatively low. Besides conventional methods of propagation, in vitro cultural methods contribute importantly for the propagation of many important and economic plants. Conventional propagation methods are very slow and propagation by tissue culture represents an important potential to effectively propagate it. Therefore, the experiment was designed to investigate the in vitro regeneration of cormlets from floral and corm segments from local ecotypes of this important plant. MATERIALS AND METHODS The corms of saffron were collected from the botanical gardens of the department of Field Crops, University of Ankara, Turkey, which were subsequently obtained from saffron producing villages of Safranbolu, the previous year. The experimental material included saffron corms, mature and immature flower bases, apical meristems etc. Surface-sterilization of corms was carried out by first scrubbing the corms gently under running tap water for 10 minutes to remove coating layer of microorganisms ubiquitously found on them. Thereafter, the corms were treated with fungicide, sulfuric acid or PPM as described below to control the development of contamination during in vitro culture of the explants. 1 minute treatment with sulfuric acid followed by sterilization with 80 % commercial bleach (ACE) for 20 min. 2.5 minute treatment with sulfuric acid followed by sterilization with 80% commercial bleach (ACE) for 20 min. 1 minute treatment with sulfuric acid followed by sterilization with 0.7 % silver nitrate for 10 min. 2.5 minute treatment with sulfuric acid followed by sterilization with 0.7 % silver nitrate for 10 min. Treatment with 3% dry or liquid fungicide. Treatment with 1, 2, 3, 4, 5 and 6% PPM in distilled water for 1 hour. The corms were also treated with or without hot water at 40, 42.5, 45 and 47.5 C before sterilizing them with 50% (v/v) commercial bleach (Axion Turkey) for 20 minutes followed by 5 rinses in sterile distilled water. Thereafter, the explants were cultured in Petri dishes® (100x10 mm diameter) /Magenta boxes containing 2 mg/L BAP- 0.5 mg/L NAA (Mirici et. al. 2005) in MS (Murashige and Skoog, 1962) regeneration medium supplemented with 3 % sucrose, 0.7% agar (Sigma agar type A), autoclaved at 121 oC under pressure of 1.2 kg/cm 2 for 20 minutes. The pH of the medium was adjusted to 5.6-5.8 with 0.1 N KOH or HCl before autoclaving. Cultures were maintained at 24 °C under white fluorescent light of 42 μmol·m-2·s-1 with the photoperiod of 16 hrs light and 8 hrs dark at 24±1°C. The explants consisted of corm buds removed from the sterilized corms, whole corms if they were smaller than 5 mm, shoots, stigma, ovary or floral bases of mature/immature flowers. RESULTS AND DISCUSSIONS Surface Sterilization Although corms were planted in the botanical gardens of the department of Field Crops for one year to avoid any chances of endogenic contamination; yet one year quarantine was not enough to do so and most of the corms remained contaminated. It was possible to remove surface contaminants by above described sterilization procedures but was not possible to remove endogenic contaminants. Only an insignificant quantity of corms could be obtained by sterilization with 100 % commercial bleach for 20 minutes. Treatment with sulfuric acid or high temperature treatments especially 47.5 oC were damaging. Most of the times it appeared that sterilization was achieved but the endogenic contaminants that were latent in the developing explants reappeared after 3-5 weeks and the experiments could not be advanced further. Air borne contaminants could be removed easily using standard sterilizing techniques. However, it is very difficult to remove endogenic contaminations even under best conditions of sterilization. We suspect spores or dominant microorganisms occurring as endogenic were resistant to disinfectants under any condition of sterilization. These findings are partially in line with Ozel et al. (2006) and Taylor et al. (1998). They found that addition of antibiotics or PPM to the culture resulted in provisional blocking of contamination for 3-5 weeks which reappeared thereafter once again. Irrespective of the treatments, an insignificant proportion of the corms could be sterilized. It was not possible to achieve significant or 100 % sterilization because of high percentage of endogenic contaminations. Therefore, it was decided to continue from those insignificant numbers of sterilized corms. Micropropagation After 3 weeks of culture, the whole corms did not show any development. However the explants consisting of eye buds showed development of single shoots (Fig. 1a). These developed into well developed shoots with leggy appearance after 6 week of culture (Fig. 1b). The shoot development was very slow, and they grew to a length of 5-6 cm after 16 weeks of culture. Further subculture on the same medium showed regeneration of one or two cormlets from the main explants after 24 weeks of culture (Fig. 1e). These corms showed dormancy. The dormancy was broken after a period of 20 weeks (44 weeks of culture) with regeneration of 2-3 cormlets per explant (Fig. 1f). The developing corms (Fig. 1g) were transferred to the pots for onward transfer to the green house (Fig. 1h). In vitro development of cormslets could not be observed from stigma or leaf explants even after a long period of culture. However, cormlet regeneration via somatic embryogenesis were achieved from mature floral bases on MS medium containing 1 mg/L BAP and 1 mg/L NAA (Fig 1c and d). Ding et al. (1979, 1981) first reported tissue culture of C. sativus using MS medium containing IAA and/or 2,4-D. It was followed by another report by Homes et al. (1987) showing development of microcorms on the surface of explants (1/8 corm fragments). Some corms germinated and produced young shoots on Gamborg Medium for minerals, MS medium for the organic part and the addition of 2,4-D and Kinetin. Ilahi et al. (1987) used corms and obtained callus that differentiated into buds on subculture on the same medium, or MS with auxin, cytokinin and coconut milk. Plessner et al. (1990) found that cytokinins and 2,4-D were utilized for the development of isolated bud explants. Himeno and Sano (1987) cultured half ovaries on different media using auxins. Similarly Fakhari and Evan (1990) obtained stigma like structures cultured from half ovaries on white medium supplemented with zeatin and NAA. Zhigang et al. (2005) reported enhancement of cell growth in suspension cultures by investigating the relationship between morphological transformation and cell growth in callus and suspension cultures of saffron cells belonging to the cell line C96 induced from Crocus sativus. They found that an unbalanced osmotic pressure between the intra cell and extra cell regions induced a large morphological transformation which affected normal division of the saffron cells. An increase in osmotic pressure caused by the addition of sucrose inhibits the vacuolation and shrinkage of cytoplasm in the cells. As the sucrose concentration increases, the total amount of accumulated biomass also increases. Besides the sucrose concentration, increased ionic strength and inoculation ratio also restrain to a large extent the vacuolation and shrinkage of the cytoplasm in the suspended cells, which results in increased biomass. In the present study we manage to produce in vitro cormlets from corm and floral base explants and our research work is in continuous to increase the numbers of cormlets per explant. We believe that any research work on saffron will help the production and conservation of this plant in Turkey, which was widely cultivated here 100 years back and is now confined to a few villages in Safranbolu. Literature Cited Abdullaev, F.I. 2002. Cancer chemo preventive and tumoricidal properties of saffron (Crocus sativus L.), Experimental Biol. Med., 227:20-25 Ozel, C.A., Khawar, K.M., Mirici, S., Ozcan, S. and Arslan, O. 2006. Factors affecting in vitro Plant Regeneration of Critically Endangered Turkish Plant Centaurea tchihatcheffii Fisch et. Mey. Naturwissenschaften.93:511-517. Deo, B. 2003. Growing Saffron – The World's Most Expensive Spice, Crop & Food Research (New Zealand Institute for Crop & Food Research), no. 20. http://www.crop.cri.nz/home/products-services/publications/broadsheets/020Saffron.pdf Ding, B., Bai, S., Wu, Y. and Fan, X. 1981. Induction of callus and regeneration of plantlets from corm of C. sativus L. Acta Botanica Sinica, 23:419-420. Ding, B., Bai, S., Wu, Y. and Wang, B. 1979. Preliminary report on tissue culture of corm C. Sativus. Acta Botanica Sinica, 21:387 (in Chinese). Fakhari, F., Evans, P.K. 1990. Morphogenetic potential of cultured floral explants of Crocus sativus L. For the in vitro production of saffron. J. Exp. Bot.. 41:47-52. Himeno, H. and Sano, K. 1987. Synthesis of crocin, picrocrocin, and safranal by saffran stigma-like structures proliferated in vitro. Agricult. 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Zhigang, G., Zhaolin, Z., Ruizhi, L. and Ying, D. 2005. Morphological Transformation of Plant Cells in vitro and Its Effect on Plant Growth. Tsinghua Science and Technology. 10:573-578.  Fig. 1. In vitro cormlet regeneration in Saffron. (a) Development of shoots from corm buds after 3 weeks of culture, (b) Growth of these shoots into multiple shoots with leggy appearance after 6 week of culture. (c-d) Cormlet regeneration via somatic embryogenesis from mature floral bases (e) Development of 2-3 corms after 24 weeks of initial culture. (f-g) Development of second generation of cormlets after 44 weeks of culture. (h) Transfer of in vitro cormlets to the pots for onward transfer to the greenhouse. ‎‮