New Cytotype of Crocus pallasii sub sp. haussknechtii from West of Iran M. Sanei, H. Rahimyan Y.M. Agayev, S. Soheilivand Department of Agronomy Agricultural Biotechnology Research Institute University of Mazandaran. Seed and Plant Improvement Institute Sari Mahdasht Road Iran P.O. Box 31535-1897 Karaj Iran Keywords: aceto-iron-hematoxylin, chromosome, Crocus sativus, evolution, karyotype Abstract C. pallasii is one of the close relatives of C. sativus. A new cytotype of this species was discovered from west of Iran (Reno valley & Islamabad-e-Gharb). For karyotype studies, Lewitsky fixative and aceto-iron-hematoxylin stain were used. Karyotype of this cytotype with 2n=12 consists of one large pair of metacentric chromosomes with a satellite in the long arm, one pair of large submetacentrics, two pairs of smaller submetacentrics and two smallest pairs, which are metacentrics. This plant cytogenetically is very similar to C. pallasii subsp. turcicus (2n=12) and also C. pallasii subsp. haussknechtii with 2n=14 chromosomes. However C. p. subsp. haussknechtii with 16 chromosomes is considerably different from these two cytotypes. It seems that during evolution new cytotype with 2n=12 derived from C. p. subsp. haussknechtii with 2n=14. INTRODUCTION The Crocus sativus aggregate (Mathew, 1977) comprises C. niveus., C. sativus., C. cartwrightianus., C. hadriaticus., C. asumaniae., C. oreocreticus., C. thomasii., C. moabiticus, and C. pallasii. One member of the Crocus sativus aggregate is C. pallasii, that's the most widespread of the saffron group species. The area in which it occurs extends from Yugoslavia to Iran and from Crimea to south Jordan, and over this immense area it varies considerably, so much so that a number of the variants have been described as separate species (Mathew, 1982). Mathew (1982) recognized four subspecies for C. pallasii as follows: C. pallasi. subsp. pallasii which is a plant of dryish scrubland, belongs primarily to the eastern Mediterranean region, occurring in the Taurus mountains and south Syria with 2n=14 chromosomes. Its Karyotype consists of three pairs of acrocentric chromosomes with small satellites on the long arms of the third pair, four pairs of metacentric and submetacentric chromosomes (Fig. 3) (Brighton, 1977). C. pallasii subsp. turcicus with narrow and pointed perianth segments can be distinguished from the other subspecies. This plant belongs to dry region of south east Turkey marking the northern edge of the Syrian desert with 2n=12 chromosomes consists of one pair of large metacentric chromosomes with small satellites on one arm, three pairs of acrocentrics and two pairs of smaller metacentrics (Fig. 3). C. pallasii subsp. dispathaceus has deep reddish-purple perianth segments and very short yellow style branches. This plant occurs in the south Turkey and north Syria with 2n=14 chromosomes. Its karyotype consists of three pairs of acrocentric chromosomes with small satellites on the long arms of the second pair, two pairs of metacentrics, a smaller pair of submetacentrics and one pair of acrocentrics. C. pallasii subsp. haussknechtii which occurs in west Iran, north east of Iraq and south Jordan, has a corm with a long fibrous neck, and very thick dark red club- like style branches. The pale bluish-liliac perianth segments are normally rounded or blunt and, in Iranian material at least, are often notched at the apex (Mathew, 1982, 1977). This subspecies has been reported with 2n=16 chromosomes. Cytotype with 2n=16 chromosomes has been collected and studied by Brighton (1977) from south west of Iran (Kazeroun and Firouzabad). Karyotype consists of three pairs of large acrocentric chromosomes with satellites on the long arms of one pair, three pairs of metacentric chromosomes and two pairs of small acrocentrics (Fig. 4). In 1989 Mathew et al. reported a new cytotype of this complex, with 2n=14 chromosomes from west of Iran (Sanandaj). Its karyotype consists of one large pair of metacentric chromosomes with satellites on short arms, two pairs of large acrocentrics, two pairs of smaller metacentrics and two pairs of small acrocentrics (Fig. 4). We found accidently two populations of this subspecies with 2n=12 chromosmes during our study of Iranian Crocus species. The objective of this paper was to introduce this new cytotype. MATERIALS AND METHODS Plant material was collected from Reno valley and Islamabad in the west of Iran, Ilam region. Corms were grown in vermiculate and kept in greenhouse. Root tips were collected in early morning and pretreated in 8-hydroxyquinolin for 4h at 24 °C. Then roots fixed in 1:1 (v/v) solution of formalin 10 % and chromic acid 1 % (Lewitsky fixative) for 36h at 4 °C, and stored in 70 % ethanol at -20 °C. For staining, the root tips were treated with 1N NaOH at 60°C for 10-13 min and stained in aceto- iron-hematoxylin for 2-3 hr at 45 °C. Meristematic region cut and macerated in cytase enzyme for 1.5 h at room temperature. Stained root tips squashed in 45 % acetic acid (Agayev, 1998). Chromosomes were measured by Micromeasure software, arranged and named in accordance with classification of Levan et al. (1964). RESULTS AND DISCUSSION A new cytotype in the Crocus pallasii complex is described in present study. Collected plant morphologically was very similar to C. pallasii subsp. haussknechtii with fibrous corm tunic which made a long neck, pale liliac almost obovate perianth segments and red club-like style branches (Fig. 1). This population has 2n=12 chromosomes included one pair of large metacentric chromosomes with satellites on the long arms, three pairs of medium size submetacentrics and two pairs of smaller metacentric chromosomes (Table 1, Fig. 3). Although comparison of chromosome type with previous works is not almost logical as mentioned authors didn't follow the widely accepted nomenclature of Levan et al. (1984). Three cytotypes of C. pallasii subsp. haussknechtii morphologically are completely similar but karyotype of C. pallasii subsp. haussknechtii with 2n=16 are different from karyotypes of two other cytotypes. Plants with 2n=14 and 2n=12 chromosomes of this subspecies have similar karyotypes. If fourth or fifth chromosome pairs of C. pallasii subsp. haussknechtii with 2n=14 chromosomes is eliminated, the resulted karyotype is considerably similar with C. pallasii subsp. haussknechtii with 2n=12. Small differences especially in satellite position can be due to small translocations in chromosome arms. It seems that these two cytotypes derived from a single ancestor during the evolution or one of them diverged from the other by a chromosomal mutation. Among subspecies of C. pallasii, there are a lot of similarities between our new cytotype and C. p. turcicus (Fig. 4), although their plant morphology is different (Fig. 2). May be C. p. turcicus and C. p. haussknechtii are synonyms and morphological differences between them could be just the environmental effect. Although proving this hypothesis needs further study of all these populations with banding or molecular cytogenetic methods. ACKNOWLEDGMENTS This work was financially supported by Agricultural Biotechnology Research Institute of Iran, and is gratefully acknowledged. We wish to thank Anthony Goode for useful helps and identification of the plant as well. Literature Cited Agayev, Y.M. 1998. Advanced squash methods for investigation of plant chromosomes. Presented Paper in Fourth Iranian Congress of Crop Production and Breeding Sciences. Isfahan University of Tech., Isfahan, Iran 25-27 Aug. p.1-20. Brighton, C.A. 1977. Cytology of Crocus sativus and its allies (Iridaceae). Plant Systematics and Evolution. 128:137-157. Levan, A., Fredka, K. and Sandberg A.A. 1964. Nomenclature for centromeric position on chromosome. Hereditas. 52: 201-220. Mathew, B. 1977. Crocus studies and its allies (Iridaceae). Plant systematics and Evolution. 128:137-157. Mathew, B. 1982. The Crocus. A revision of the genus Crocus (Iridaceae). B.T. Bats ford Limited. London. Mathew, B., Brighton, C.A., Baytop, T. 1979. Taxonomic and cytological notes on Asiatic Crocus. Notes from the Royal Botanic Garden, Edinburgh. 37(3):469-473. Table 1: Karyotype characteristics of C. pallasii subsp. haussknechtii(2n=12) Typ Long arm Chr. Short arm. Total length Arm ratio Satellite e (µm) 4.68 ± 1.26 ± 0.99 1 m 5.94 ± 0.28 10.62 ± 0.46 0.18 0.02 ±0.08 2.93 ± 2 s.m 5.87± 0.22 2.01 ± 0.07 7.89 ± 0.27 _ 0.10 3 s.m 5.27 ± 0.20 1.96 ± 0.07 0.27± 7.23 2.69± 0.05 _ 2.25 ± 4 s.m 4.17 ± 0.17 1.86 ± 0.06 0.19±6.03 _ 0.10 1.24 ± 5 m 3.58 ± 0.16 2.06 ± 0.08 0.21 ± 5.64 _ 0.09 1.55 ± 6 m 3.12 ± 0.16 2.02 ± 0.07 0.20± 5.15 _ 0.07 Total 42.58 ± 2.15 m = metacentric s.m = submetacentric Fig. 1. Flower and corm of Crocus pallasii subsp. haussknechtii (2n=12) Fig. 2. Flowers of Crocus pallasii subsp. turcicus (2n=12) Fig. 3. Methaphase chromosomes of Crocus pallasii subsp. haussknechtii (new cytotype) and its ideogram New cytotype of C. pallasii haussknechtii (2n=12) C. pallasii turcicus (2n=12). Source: Brighton, 1977. C. pallasii haussknechtii (2n=14). Source: Mathew et al., 1978. C. pallasii haussknechtii (2n=16). Source: Mathew et al., 1978 Fig. 4. Comparing of different cytotypes of Crocus pallasii subsp. haussknechtii and C. pallasii subsp. turcicus .