C 1999 The Japan Mendel Society Cytologia 64: 181-196, 1999 Determination of Chromosome Number of Kuwaiti Flora I. G. A. Malallah and G. Brown Department of Biological Sciences, Faculty of Science, Kuwait University, Kuwait. Accepted February 12, 1999 Summary This paper deals with the chromosome numbers (somatic or sporophytic and gametic) of some wild species of the Kuwaiti flora. All species were collected from the wild except Plantago lanceolata, which was cultivated. Some values are reported for the first time. The species and their respective chromosome numbers are as follows: Anthemis deserti 2n=18, Atractylis carduus 2n=20, Calendula arvensis 2n=36, 44, Koelpinia linearis 2n=54, 36, Launaea capitata 2n=18, L. mu- cronata 2n=16, Picris babylonica 2n=10, Reichardia tingitana 2n=16, Senecio glaucus 2n=20, Sonchus oleraceus 2n=32, Astragalus hauarensis 2n=48, A. schimperi 2n=16, Lotus halophilus 2n=14, 28, Medicago laciniata 2n=16, Plantago amplexicaulis 2n=10, P. boissieri 2n=10, P. cilia- ta 2n = 10, P. coronopus 2n= 10, P. lanceolata 2n= 12, P. ovata 2n =8. Karyotype analysis of P. baby- lonica, R. tingitana, P. boissieri, and P. ciliata is shown. The chromosome set is not only one of the most characteristic attributes of a species, but also carries the bulk of essential information for controlling its organization and metabolism. Therefore, variations in number, size and form of the chromosomes between genera and related species, and sometimes between populations of the same species, are of importance for classification and certain evolutionary aspects (Sato 1962). Such variations represent different karyotypes (Jackson 1971). Numerical analysis of chromosomes is one of the processes in assessing the genomic status of a species. Chromosome numbers of species can provide useful information for various fields of re- search including karyotaxonomy, genetics, cytogenetics, plant breeding, ecology, biogeography and molecular biology. A number of workers have been involved in reporting the chromosome numbers of wild and cultivated plant species (Darlington and Wylie 1955, Bolkhovskikh et al. 1969, Love and Love 1974, 1975, Moore 1973a, b, 1977, Meikle 1977, Beuzenberg and Nair 1983, Goldblatt 1981a, b, 1985, Webb and Beuzenberg 1987, Majovsky and Murin 1987, Rao and Chandel 1991). On the basis of such information it has often been possible to interpret the genetic interrelationships existing within species (Berdahle and Barker 1991, Brochmann 1992, Stepankova 1993). Halwagy and Macksad (1972) give a historical account of the exploration of the Kuwaiti flora, but no infor- mation on chromosomal aspects has been reported. Indeed, there is a complete lack of knowledge regarding the karyology of wild plant species in Kuwait (Malallah et al. 1996). The main aim of this paper is to provide information on the chromosome number of various species of the Kuwaiti flora. In this initial study, 20 species from three different families were se- lected, and their mitotic and meiotic chromosome numbers determined as well as the mitotic index, karyotype analysis of 4 species is also considered. Materials and methods 20 species from three families, Asteraceae, Fabaceae, and Plantaginaceae were selected for this study. Identification of the species was checked by Mrs. M. Al-Doseri, Biological Sciences- University of Kuwait. Nomenclature and synonyms are in accordance with Boulos and Al-Dosari (1994), Daoud (1985) and Al-Rawi (1987). Seeds of each species were collected from at least three different plants growing at various lo- 182 G. A. Malallah and G. Brown Cytologia 64 calities in Kuwait during November to April 1993 and 1998. They were germinated in a controlled growth chamber for 12 h dark at 16•Ž and 12 h light at 22•Ž on moist filter paper enclosed in Petri dishes. Actively growing root tips were excised and fixed in Carnoy's fixative (1 : 3 acetic acid alco- hol) until ready for use. For slide preparation, excised roots were washed with distilled water for 4- 5 min, hydrolyzed in 1N HCl for 10-12 min at 60•Ž and finally rinsed with distilled water for 4- 5 min. The roots were stained in 1% aceto-carmine for microscopic analysis. Some three-day old seedlings were treated with 0.05% colchicine for 4 h or 20 h cold water treatment at 50•Ž in order to have c-metaphases. Young flower buds were collected and kept in Carnoy's fixative until further use. After discard- ing unwanted tissues, anthers were squashed in a drop of aceto-carmine stain and covered with a clean cover glass. Microscopic analysis was carried out with an Olympus BH-2 photomicroscope. Micrographs were taken when necessary from temporary preparations. Slides were made permanent by removing the cover slip with dry ice techniques followed by dehydration of the slides in increas- ing alcohol concentrations. Several well-spread metaphases were counted to confirm the validity of the chromosome number and for karyotype analysis. The mitotic index was calculated by recording the stage of every nucleus present in a field of view at 400 •~ magnification. When expressed as a percentage, the relative amount of time nuclei spent at each mitotic stage can be determined. Data on general distribution and various ecological aspects of individual species were taken from the lit- erature, although we have added our own observations when more appropriate. Karyotype analysis For diploid karyotype analysis, a minimum of 5 root tip cells from each case was analyzed. Cells in which the chromosomes were well separated were photographed at high magnification. Photo-ideograms were prepared from the photographs by cutting out individual chromosomes and arranging them in a descending order of their length. On the basis of gross morphology and centromere position, the chromosomes were grouped to form homologous pairs. Averages of the paired chromosomes were taken for total length, arm ratio and total volume. The total length was ascertained by adding the length of all chromosomes in the karyotype. The ratio of the long arm (L) to the short arm (S) was used to identify centromere posi- tion for each chromosome. The nomenclature system of Levan et al. (1964) was used to determine the chromosome types as median (m), sub-median (sm) or sub-terminal (st). The chromosome vol- ume was determined from the metaphase plate, assuming the chromosome as cylindrical structure by the formula ƒÎr2h, where r and h represent the radius and the length of the chromosome respec- tively. The total volume is determined by adding the volumes of all chromosomes. Results and discussion The mitotic and meiotic chromosome numbers and the mitotic index of each species are listed in Table 1 while the corresponding data from the literature are included in the text. Further details on morphological and ecological aspects of each species in Kuwait, are available from Al-Rawi (1987) and Boulos (1987). The rate of mitotic division in the indicated species has not been investi- gated before. In some systems, the mitotic index or the proportion of cells in visible stages of divi- sion directly reflects the relative duration of mitotic stages (Dyer 1979). The procedure performed in the present work demonstrates an asynchronous fashion, and cannot therefore provide an ab- solute value for the duration of the individual stages, or the duration of the whole cycle. Further work is required to clarify. However, the mitotic index mean gives an indication of that mitosis oc- cupied a typical value for the total time (Brown and Bertke 1974). The life cycle of annual wild species in Kuwait is very short and the germination starts just several days after two or three show- ers of rain in the beginning of the season and develops into full plants in very short time period in- 1999 Determination of Chromosome Number of Kuwaiti Flora I. 183 Table 1. Chromosome number and mitotic index of some wild plant species in Kuwait dicating that the main factor affecting the germination is the rain. The question is whether the life span of the wild species could be controlled or not and the duration of the cell cycle are the subjects of future work. ASTERACEAE Anthemis deserti Boiss. Syn.: A. melamopodina subsp. deserti var. transiens Eig loc.cit.; A. diffusa Salzm. ex DC. Geographical distribution: Saharo-Arabian. Kuwaiti data: Found in many localities particularly common at Al-Sabiyah. Flowering time: Febru- ary-April. Literature data: No information. Comment: According to the literature, this species has not been investigated cytogenetically. This study therefore provides the first count of the chromosome number and the relative duration of mi- tosis. An interesting observation was that most cells were 2n=18, while a few others were 2n=24. A possible explanation is that some capitula had seeds of another species attached to them. We therefore consider 2n=18 to be the correct value. Atractylis carduus (Forssk.) Syn.: A. flava Coss. and Dur.; Centaurea carduus Forssk. Geographical distribution: Saharo-Arabian. Kuwaiti data: Found in many sandy localities. Flowering time: March-April. Literature data: 2n=20 (Waisel 1962); A.flava 2n=20 (Brullo et al. 1990). Comment: The somatic number of 2n=20 is the third published karyological study for this species and the first undertaken on Kuwait material. This mitotic chromosome number was the same from the three different localities in Kuwait and which is consistent with the findings from the other countries indicating the high stability of the diploid number. Accordingly, the gametic number 184 G. A. Malallah and G. Brown Cytologia 64 n=10. Calendula arvensis L. Syn.: C. sancta L.; C. persica C. A. Mey.; C. aegyptiaca Desf. Geographical distribution: Mediterranean, Irano-Turanian, and Saharo-Arabian. Kuwaiti data: It is an annual herb, which can be found in many localities in sandy places. Flowering time: February-April. Literature data: 2n=22 (Aparicio et al.