CHROMOSOMAL STUDIES ON FOUR IRANIAN CROUS ()

H. Ebrahirnzadeh, A. Saboora, M. R. Noori-Daloii and S. M. Ghaffari

Ebrahimzadeh, H.; Saboora, A; Noori-Daloii, M. R. and Ghaffari, S. M. 1998 05 25: Chromosomal studies on four Iranian species (Iridaceae). -Iran. Joum. Bot. 7(2): 179-192. Tehran.

Four species of Iranian Crocus were chromosomally investigated and their karyotypes were determined: C. sativus (2n=24), C. cancellatus subsp. damascenus (2n=8A), C. gilanicus (2n=24) and C. speciosus subsp. speciosus (2n=12). On the meiotic studies of pollen mother cells in Crocus sativus unbalanced distribution of chromosomes in two poles of cells and chromosomic lagging were frequently observed. Karyological studies on the meristematic cells of neoformed root tips in explant of saffron corm showd cells with 2n=16 chromosomes.

Hassan Ebrahimzadeh and Azra Saboura, Department of Biology, Faculty of sciences, University of Tehran, Tehran, Iran. -Mohammad Reza Noori-Daloii, Department of Biochemistry, Faculty of Medicine, Medical Sciences University of Tehran, Tehran, Iran. -SeyyedMahmoud Ghaffari, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. 4-i1 ~\;y")15J..:.,.j})1) !J""')J". ;)).Y' ,fJj.Y'J"?)a; jI01f.-10Ipj~.J)~

);) 8A 4(Corcus sativus) 'of J)y.. 01pj);) n 4 J".'.r. l,.,.~.J If.-I);) 2n .J.,.;, ~ );)n 4(CroCUScancellatus subsp. damascenus) ~;) ~.Jf.-j ~ 0ipj (Crocus 4~j ~.J.r'-j ~j 01~j);) ,r J (Crocus gilanicus) 0~ 01~j

);) 0;) J ~b );)lA .s4J ..,L j~ ~ !J""')J".);) .;)Yo speciosus subsp. speciosus) ~ ,fJj.Y'J..? ~h J J..,L ~);) '+oJj.Y'J..?J;)~L; tPy 4'ofJ)y..01pj .sl,.,.~..:....:sl~....Jai);) ..l.:.~y.sl,.,.~) ~yly")15 !J""')J"..J.,.;, •.u.~ .)IJI) .;)b0L.:..i1)2n=164 .f-4J..,L ;)Y>:-J'ofJ)y.. 01pj IRAN. JOURN. BOT. 7 (2), 1998 Crocus in Iran 181

INTRODUCTION

Crocus species are perennial, grassy and meters above s. 1. Distribution of ornamental from lridaceae family, C. gilanicus is in N. and NW. Iran, in the flowering in early spring or early autumn, provinces of Azerbayejan and Gilan. according to the species. The number of Mathew and Brighton (1976) reported species are reported 80 to 100 (Mathew, 2n=24 for the species. 1982). Their distribution centre is mainly C. cancellatus subsp. damascenus Turkey and Greece. Mobayyen (1974), distributes to the west of Iran and 2n=8, Wendelbo and Mathew (1975) and 10, 12 were reported for it (Mathew, 1982). Wendelbo (1977) have reported eight wild C. speciosus is one of the most beautiful and cultivated Crocus species in Iran. ornamental species in the garden and it Brighton (1977a, b), and Brighton, Mathew includes three subspecies, two of which are and Marchant (1973) showed that Crocus is endemic to small areas of Turkey, and the a highly complex with a wide range third, namely subsp. speciosus occurs in a of chromosome numbers (2n=6-64) and a much wider area of Iran (Caspin region considerable variation in karyotypic extending to just East of Gorgan). Its morphology (Rudall Paula, Owens and chromosome number was reported 2n= 12 Kenton 1984). On the other hand, some (Brighton, 1983; Mathew, 1982). species reveal distinct constancy in In first cytological studies on C. sativus characteristics of their karyotype. that were taken up in 1926 by Himmerbaur This report examines chromosomal the chromosome number was determined particularities in Crocus sativus and three 2n=24. Later on, other researchers wild species (c. gilanicus, C. cancellatus confirmed the first report (Brighton, 1977 and C. speciosus) from Iran. b; Karasawa, 1933, 1943; Morinaga and Among the above mentioned species, Fukushima, 1931; Pathak, 1940; Pogliana C. gilanicus is endemic of Iran. It is a small and Grosso, 1971). Karasawa (1933, 1940, white flowered species, flowering in 1943) reported that the cultivated October in grazed pastures or at the edge C. sativus was an auto triploid with of beech forests, at an altitude 1500-2400 2n=3x=24 chromosomes. He also, 182 H Ebrahimzadeh & al. IRAN. JOURN. BOT. 7 (2), 1998

Table 1. Chromosome numbers and origins of the Crocus collections studied.

Taxon Cytology Collector Locality 2n

no. C. cancellatus Herb. subsp. damascenus

(Herb.) Mathew 301 Saboora et al. [ran: Arak 8 .. 303 Saboora et al. [ran: Golpayegan 8 C. speciosus M. B. 801 Saboora et al. [ran: Golestan forest 12 C. gilanicus Mathew 601 Saboora et al. Iran: Syah-Bisheh 24 C. sativus L. 901 Saboora et al. Iran: Cultivated (in vivo) 24 - 902 Saboora el at, :ran: Cultivated (in vitro) 16

reported 2n=2x=16 and 2n=5x=40 for Corms of Crocus sativus were collected C. sativus. Noori-Daloii (1975), from a farm situated in Gonabad and, three Aghamohammadi (1976) and Estilai and wild species, including C. cancellatus subsp. Aghamohammadi (1997) have reported damascenus from two populations in Arak that Iranian saffron has 2n=24 and Golpayegan, C. speciosus subsp. chromosomes. Ghaffari (1986, 1991) speciosus from Golestan forest on the road obsdrved that C. sativus is an toward Bojnoord and C. gilanicus from autotriploid which fromed up to eight Siah-Bisheh (Table 1). trivalent groups of chromosomes at both Root tips were collected and pre-treated diakinesis and metaphase stages during in 0.002 m 8-hydroxyquinoline for seven meiosis, Furthermore, presence of anomaly hours at a temperature of 25-30°C. They that occurring in segregation of were then fixed in Carnoy and Piennar's chromosomes during meiosis produces a fixator for 24 hours (Philips, 1981) and variety of microspore with gametic stored in 70% ethanol at 4°C. After chromosome numbers ranging from 8 to 16. staining and squash by 1% Fe-acetocarmine (Philips, 1981), suitable slides were MATERIALS AND METHODS prepared. Average of lenght and diameter, and arm ratio (long: short) were computed The plant materials wene investigated in for each chromosome and they were autumn when root formation is active. categorized in tables 2-5. IRAN. JOURN. BOT. 7 (2), 1998 Crocus in Iran 183

Table 2. Morphometric characteristics of C. Table 3. Morphometric characteristics of C. cancellatus subsp. cancellatus chromosomes. speciosus subsp. speciosus chromosomes.

Number of Tollil I Arm raUo Dtarneter Number of Total Arm ratio Diameter chromosome pair lenght (p.) (long: short) (p.) ehrnmosorue pair lenghl (p.) (long: short) (p.) -- 1 12 1.37 2.5 3.97 2.37 I 8.50 2 12 1.22 2.5 2 7.82 4.99 2.42 3 11.87 1.43 2.5 4 10.75 1.53 2.5 2.26 3 6.75 4.06 5 9 I 1.52 2.5 7 1.08 2.5 4 5.35 I 2.33 I 2.18 6 2.31 average 10.44 1.36 2.5 average 7.1 3.84 I

Table 4. Morphometric characteristics of Table 5. Morphometric characteristics of chromosomes of C. gilanicus. C. sativus chromosomes.

Number of Total Arm ratio Diameter Number of Total Arm ratio [Diameter chromosome pair lenghl (p.) (long: short) (p.) - chromosome pair lenght (p.) (long: short) (p.) 1.11 1.75 1 19 1 6.41 3.40 0.61 2 10 1.33 2 3 9.5 5.3 2 2 5.80 2.22 0.61 2 4 9 2 3 4.58 1.07 0.5 5 8.5 3.66 2 6 7.5 2.8 2 4 3.66 1.10 0.61 5 2 7 6.5 5 3.62 1.2 0.61 8 6 3 2 9 5.5 1.2 2 6 3.58 2.56 0.61 5 233 2 10 7 3.05 1.89 0.61 11 5 4 1.5 4.5 2.5 2 1.12 0.61 12 I 8 2.14 - I average 8 T 2.84 1.94 average 4.11 1.82 0.6 J

In order to study the changes occurring were observed in pollen mother cells. in cells of corm explants of C. sativus with Immature flower buds taken from the regard to chromosome number, segments underground corms, and then fixed in of callus and neoformed roots were stained Piennar's solution for 24-48 hours at 4°C. with the same method. After washing with distilled water, they Meiotic chromosomes of Crocus sativus were stored in 70% ethanol at 4°C. The 184 H Ebrahimzadeh & al. IRAN. JOURN. BOT. 7 (2), 1998 anthers were then stained and squashed in forest, reveals 2n=12. This species shows a 2% Fe-acetocarmine. distinctive karyotype of two pairs of metacentric chromosomes with small RESULTS satellites on the one pair, three pairs of submetacentric and one pair of smaller The results obtained from karyological metacentric chromosomes (Fig. 1b, 2b). studies in Crocus species are summerized as Morphometric characteristics of C. specious follows: are presented in Table 3.

Crocus cancellatus subsp. damascenus C. gilanicus The collections of C. gilanicus from Mitotic studies on meristematic cells of Syah-Bisheh in Mazandaran province have root tips in collected specimens from 2n=24, the karyotype consisting of two Sefidkhany plain of Arak and Golpaygan pairs of metacentric, one pair of smaller had no distinct difference in chromosome metacentric, one pair of submetacentric, number, 2n=2x=8A. Usual karyotype of one pair of acrocentric, four pairs of these populations consisted three pairs of middle-sized acrocentric and three pairs of acrocentric and one pair of smaller smaller acrocentric chromosomes. There acrocentric chromosomes. There are are satellites on the short arms of one pair extremely small satellites on the long arms of acrocentric chromosomes (Fig. Ic, 2c). of the fourth pair which were faintly Morphometric characteristics of stained and observable in some specimens chromosomes of C. gilanicus are presented with difficulty (Fig. la, 2a). Morphometric in Table 4. characteristics of C. cancellatus, eytotype 2n=8A, are summerized in Table 2. C. sativus Karyological studies on cultivated C. speciosus subsp. speciosus C. sativus (saffron) revealed an The cytological studies of this subspecies autotriploidy with 2n=24 chromosomes. collected from the margin of Golestan The karyotype of which consists two IRAN. JOURN. BOT. 7 (2), 1998 Crocus in Iran 185 triplets of acrocentric, three triplets of mitotic metaphase shows 2n=16 (Fig. 2e). metacentric, two triplets of smaller acrocentric and one triplet of smaller DISCUSSION metacentric chromosomes. There are satellites on the large arms of the second C. cancellatus subsp. damascenus (2n=8) as groups of acrocentric chromosomes (Fig. compared with other species which are 1d, 2d). Characteristics of chromosomes of investigated in this paper has enjoyed less C. sativus are presented in Table 5. antiquity for smaller basic chromosomal Meiotic studies on pollen mother cells in number (x) and chromosome humber cultivated C. sativus showed special (Bidault, 1971), existence of acrocentric anomalies of auto triploid plants. The chromosomes and small satellites (Pathak, segregation of chromosomes and their 1940). migration to two poles of cell in anaphase I Constant difference in size of the same is occurred at random and has caused the or different pairs in a species may be due derangements. In addition, retardation of to the stage of evolution of the particular some chromosomes and chromosome species in the genus, the most primitive elimination are also observed in some cells. ones possessing large, and the advanced The result of this process is unbalanced ones possessing minute satellites [Pathak, distribution of chromosomes in two poles 1940]. of cell (Fig. 3). Specimen of C. speciosus subsp. Staining and counting of chromosomes speciosus, collected from the margin of in callus cells and neoformed root tips of Golestan forest showed a chromosome explant shows that in the basic medium LS number 2n=12. Brighton (1983) reviewed (Linsmaier and Skoog, 1965) which this subspecies and introduced seven contains 1 mgllit each of both hormone cytotypes. In these studies, the collections NAA and BAP, there are cells with of North of Iran had 2n=12 with a different chromosome numbers in the distinctive karyotype of three pairs of large meristematic zone of neoformed roots. metacentric chromosomes with small Chromosome counting of these cells in satellites on one pair, and three pairs of 186 H Ebrahimzadeh & al. IRAN. JOURN. BOT. 7 (2), 1998 submetacentric with small satellites on the Cariola, 1982; Ghaffari, 1986, 1991; long arms of one pair Karyotype of the Karassawa, 1933, 1943; Noori-Daloii, 1975; above specimen and those investigated in Pathak, 1940; Pogliana and Grosso, 1971]. this study are fundamentally similar. Only Reported karyotype was similar to in the latter case one pair metacentric karyogram of C. sativus that was reported chromosomes is replaced by one pair of from central region of Iran by Brighton submetacentric that could be caused by (1977 b). exchange of chromosome pieces. Investigation of the stage of micros pore Karyotype of C. gilanicus shows 2n=24 formation from pollen mother cells reveals which consists of two distinctive groups of cytologically abundant abnormal states. chromosomes i. e., large and small. These During the meiosis, disorderly distribution two types of chromosomes are derived of chromosomes, occurence of laggared probably from similar karyotypes with the chromosomes and, in some cases, origin of polyploid, smallers resulting from chromosomal deletion is observed. differential loss of the large chromosomal Therfore, after the completion of meiosis, pieces. Translocation of chromosomal the micros pores are with different pieces is also another probability that chromosome number that their caused decrease of lenght of donor germination power are incomplete and a chromosomes and increase of lenght of few number of them remain alive. Ghaffari receptors. An example is karyotype of (1986, 1991) reported existence of garnets Aloinae that consists of approximatly equal that have 8-16 chromosomes. This state number of small chromosomes and large caused that garnets with respect to presence chromosomes [Stebbins, 1989]. of genes have been unbalanced and The results obtained from mitotic and consequently sterile. One of the reasons for meiotic studies in C. sativus show that it is the sterility of C. sativus were mentioned a autotriploid plant (2n=3x=24) its autotriploidy and apearance of conforming with previous reports anomalies during formation of micros pores [Aghamohammadi, 1976; Brighton, 1977b; (Chichiricco and Grilli Cariola, 1982, 1986; Chichiricco, 1984; Chichiricco and Grilli Chichiricco, 1984) and megaspores IRAN. JOURN. BOT. 7 (2), 1998 Crocus in Iran 187

(chichiricco, 1987). Tehran. In the mitotic studies on the corm Bidault, M. ]971: Variation et Speciation explant of C. sativus (in vitro), we observed Chez Les Vegetaux Superieurs. Notions reduction of chromosome number (2n=16). Fondamentales de Systematique Unfortunately, karyotyping of these cells Moderne. -Paris. were difficult and existence of two series of - B. Mathew and C. J. Marchant, 1973: chromosomes (diploidy) were not Chromosome counts in the genus confirmed. Whereas various examinations Crocus (Iridaceae). Kew Bull., 28, for obtainment of plants that can produce 451-464. the germinative seeds were not successful Brighton, C. A., 1977 a: Cytological [Aghamohammadi, 1976; Chichiricco, 1986; prolems in the genus Crocus, II. Crocus Ghaffari, 1991]. cancellatus aggregate. -Kew Bull, ."2-33-45 ACKNOWLEDGMENTS - , ]977 b: Cytology of Crocus satuvus L. (Iridaceae) and its allies. -PI. Syst. Evol., We are grateful to the National Research 128: 137-157. Center for Genetic Engineering and - , 1983. A detailed study of Crocus Biotechnology for financial support during speciosus and its ally C. pulchellus the course of this research. (Iridaceae). -PI. Syst. Evol., 142, 187-206. REFERENCES Chichiricco G., ]984. Karyotype and meiotic behavior of the triploid Crocus Aghamohammadi, Z. 1976: Investigation of sativus L. -Cariologia, 37(3): 233-239. vegetative propagation, establishment of 19R7: Megasporogenesis and variation by mutagen agents and study development of embryo sac in Crocus of pollen stainability and pollen sativus L. -Cariologia, 40(1-2), 59-69. germination in relation to sterility of - and M. Grilli Cariola, 1982: Germination saffron (Crocus sativus L.). Thesis of and variability of the pollen of Crocus MS. -Faculty of Sciences, University of sativus L. -G. Bot. ItaI., 116: 167-173. & 188 H Ebrahimzadeh al. IRAN. JOURN. BOT. 7 (2), 1998

r-----+ 10 }l

(b)

- :!.f I ••

II " I'"'6

10 }l

., ( d 1 'a. ,• l( t'l ,, 1\ )t, 11> " :n

!'--- 10 V

Fig. 1. Karyotype of (a) Crocus cancellatus subsp. dcmascenus (2n=8A); (b) C. speciosus subsp, speciosus (2n=12); (c) C. gilanicus (2n=24); (d) C. sativus (2n=3x=24). IRAN. JOURN. BOT. 7 (2), 1998 Crocus in Iran 189

Fig 2. Mitotic chromosomes :Jl CI()Cl'~ suecies: (~) C cancellatus subsp. damascenus; (b) C. speciosus subsp. speciosus; (c) C. r:r;[ tnicus (J) C. sativus (e) C. sativus, metaphase ;

mc-isterr ':It;:: re1ls of neoforrned root ti:~~ 0 (,;[ID zxnlar; t, 2n= 16. 190 H Ebrahimzadeh & al. IRAN . .TOURN. BOT. 7 (2), 1998

Fig. 3. Meiosis in pollen mother cells (If Crocus sativus: (a) methaphase I, 2n=24; (b) early anaphase 1, migration of chromosomes to two poles of cell: (c) segregation in the form of 11-3-10; (d) 9-7-8 segregation; (e) 8-4-12 segregation; (f) stage of tetrad, formation of fcur cells with one or two nucleus; separated micros pores with two nucleus, is also observed, IRAN. JOURN. BOT. 7 (2), 1998 Crocus in Iran 191

-, - 1986: Crocus sativus pollen germination Plantarum, vol. 18: 100-127. and pollen tube growth in vitro and Mathew B., 1982: The Crocus. A revision after intraspecific and interspecific of the genus Crocus (Iridaceae). pollination. -Can. J. Bot.: 64, 2774-2777. -London, Batsford Ltd. Estilai A., and Aghamohammadi Z. 1977: - and C. A. Brighton, 1976: Notes on Pollen stainability and pollen Crocus gilanicus. -Iran. Journ. Bot., 1: germination in relation to sterility of 19-21. saffron (Crocus sativus L.). -Bull. Fac. Mobayyen S., 1974. Plants of Iran, Vascular Sci. Tehran. Univ. 9(1), 10-15. Plants. vol. 1, Gymmnosperms, Ghaffari, S. M. 1986. Cytogenetic studies of Monocotyledons. -Tehran, Tehran cultivated Crocus sativus (Iridaceae). University. -PI. Syst. Evol.: 153, 199-204. Morinaga T. and E., Fukushima 1931: - , 1991. Cytologically studies on C. sativus Chromosome numbers of cultivated L. from Iran. Thesis, -Institute of plant. III. -Bot. Mag. (Tokyo), 45, 531: Biochemistry and Biophysics, Tehran 140-145. University. Noori-Daloii M. R., 1975: Cytogenetical Himmerbaur W., 1926: Zur studies of Crocus sativus L. and Entwicklungsgeschichte von Crocus investigation of its planting possibility sativus L. Festschrift, Tschirch Taunitz, in other region of Iran. Thesis of MS, Leipzig, 335-350. Faculty of Sciences, -Tehran University. Karasawa K., 1933: On the triploidy of Pathak G. N., 1940: Studies in the cytology Crocus sativus L. and its high sterility. of Crocus. -Ann. Bot, new series 4: -Jap. J. Genet., 9: 6-8. 227-256. - , 1940: Karyological studies in Crocus. II. Philips R. L., 1981: Plant cytogenetics in -Jap. J. Bot., 2: 129-140. staining procedure. U. S. A, Clark G. - , 1943: Karyological studies on Crocus. (ed). -Wilkins. III. -Jap. J. Bot., 12: 457-503. Pogliani M. and F. D. Grooso, 1971: Studio Linsmaier G. M. and F., Skoog, 1965: cariologico di Crocus sativus. -Inform. Organic growth factor requirements of Bot. Ital. 4: 25-29. tobacco tissue cultures. -Physiologia Rudall Paula J., S. J. Owens and A. Y. 192 H. Ebrahimzadeh & al. IRAN. JOURN. BOT. 7 (2), 1998

Kenton, 1984: Embryology and breeding Arnold, London. systems in Crocus (Iridaceae): A study Wendelbo P. and Mathew B., 1975: in causes of chromosome variation. -PI. Iridaceae in K H. Rechinger Fl. Iranica. Syst. Evoi. 148: 119-134. no. 112. -Graz. Stebbins G. L., 1971: Chromosomal - , 1997: Tulips and Irises of Iran, their evolution in Higher Plants, Relatives. -Botanical Institue of Iran, Contemporary Biology Series. -Edward Tehran.