Annals of RSCB Vol. XVII, Issue 2/2012

Transcription

1 CYTOGENETIC STUDIES REGARDING TWO SPECIES OF INULA FROM THE ROMANIAN FLORA: INULA HELENIUM L. AND INULA ENSIFOLIA L. Marinela Afemei 1, C. Tudose 1 *, Gabriela Voichita 2 1 UNIVERSITY AL. I. CUZA IAŞI, FACULTY OF BIOLOGY 2 BIOLOGICAL RESEARCH INSTITUTE IASI Summary Nowadays, karyotyping and the morphological study of the mitotic chromosomes are compulsory stages for the complete characterization of a given species. In the present paper are described the karyotypes and the morphological traits of the chromosomes of two species of Inula: Inula ensifolia L. and Inula helenium L. We have used achenes of the two species. After germination the roots were prefixed in 0,2% colchicine, fixed and stained according to Feulgen method. We made microscopic slides by the squash method. The detection of the homologous chromosomes and the determination of their position in karyotype was carried out according to the method of Levan et al., The karyotype of Inula ensifolia L. (2n=16) is represented by 7 pairs of median type (m) and a pair of submedian chromosomes (sm). All studied specimens were diploid. With I. helenium, we could establish only the number of diploid chromosomes, 2n=20, and we could identify a pair of submedian chromosomes with satelites. Also no polyploidisation phenomena were observed. As we know, this paper is the first report of karyological analysis of these species in Romania and our results are convergent with the data reported by foreign authors. Keywords: Inula helenium L., Inula ensifolia L., karyotype. *cristian.tudose@uaic.ro Introduction The morphological description of the mitotic chromosomes, as well as their arrangement in a karyotype, are nowadays compulsory for the complete characterization of a given species. In the present paper we are studying the morphological traits of the chromosomes of two species of Inula: Inula ensifolia L. and Inula helenium L. Consulting the specialty literature, we were surprised to see that data regarding the morphological description of these species chromosomes are scarce, even some of them are well known medicinal plants. Previous studies (Fedorov, 1969; Kuzmanov, 1993; Goldblatt and Johnson, 2000) determined in the somatic cells of Inula ensifolia L. a chromosome number 2n = 16, all the studied specimens being diploid. We found a single scientific paper (Kamari et al., 2008) referring to the morphology and typology of the chromosomes of this species, but for a triploid variety (2n = 24 =3x = 16m + 4m SAT + 2sm SAT + 2t SAT ). Similarly, for Inula helenium L., scientific papers (Rutland, 1941; Morton, 1977; Ma et al., 1985; Kokubugata et al., 2002) report 2n = 20, being described also some polyploid specimens with the base number x = 10. We found only two scientific papers referring to the morphology and typology of the chromosomes of this species, but with different results: 2n = 20 = 16m + 2sm + 2sm SAT (Ma et al., 1985) and 2n = 20 = 14m + 2sm + 2sm SAT + 2st (Kokubugata et al., 2002). The lack of complete cytogenetic data in the international specialty literature and the absence of similar studies in Romania motivated us to study these two species as a continuation of our previous cytogenetic studies in other species of Inula (Afemei et al., 2012). 192

2 Material and methods We have used achenes of the two species harvested from mature plants growing at Valea lui David, Iassy County for I. ensifolia and from Miclăuseni, Iassy County for I. helenium. After germination the roots were prefixed for two hours in 0,2% colchicine, fixed and stained according to Feulgen method (Tudose., 1976). We made microscopic slides by the squash method. Metaphases with well spread chromosomes were microphotographed using the Nikon Eclipse 600 microscope digital camera with the 100x immersion oil objective. The chromosomes were measured using a micrometer objective scale. Their length was expressed in microns, according to a micrometer scale. For each chromosome, the following characteristics were studied: arm length (q - long arm and p - short arm), total chromosome length (p + q), the difference between arms (q p), presence of satellites (s) and primary constrictions (h); arm ratio(q/p), relative length and centromeric index. The detection of the homologous chromosomes and the determination of their position in karyotype were carried out according Levan method (Levan et al., 1964). Results and discussions Morphological study of Inula helenium L. chromosomes The analysis of the well exposed chromosomes, but in metaphases with two or more superpositions, allowed us to confirm the diploid number of this species as being equal to 20, then 2n=20, in accordance with the data found in the speciality literature (Kokubugata et al., 2002), but didn t allow us to compose a karyotype that meets all the requirements. Without performing all the measurements, we may notice from the analyzed metaphases two categories of chromosomes: medians (m) and submedians (sm). We appreciate that the typology of the chromosomes is closer to the researches made by Ma et al., in 1985, than those of Kokubugata et al., in Using the classical cytogenetic techniques, we succeeded to identify secondary constrictions that represent sateliferous peduncles and satelites on a pair of submetacentric chromosomes (fig.1), thus being in accordance with the data from the speciality literature (Ma et al., 1985 and Kokubugata et al., 2002). Figure 1: Metaphase - Inula helenium L. (2n=20) the satelites are marked on the short arms of a pair of sm chromosomes Because we identified only two types of chromosomes and keeping in mind Stebbins classification, we can also state the fact that 193 the karyotype of Inula helenium L. shows a high degree of symmetry, proving that during its evolution didn t occur important

3 structural rearrangements. We must add to this statement the fact that we couldn t identify any case of polyploidy in all the studied specimens. To finalize our study on the morphometry and typology of Inula helenium L. chromosomes, we will continue our research on a large number of metaphases. Morphological study of Inula ensifolia L. chromosomes The analysis of the well exposed chromosomes, with less than two superpositions per metaphase, allowed us to establish the diploid number to be equal with 16, so that 2n=16, in accordance with the data reported in the available scientific literature (Fedorov, 1969; Kuzmanov, 1993; Goldblatt and Johnson, 2000) and allowed us to perform the karyotype of this species (figures 2 and 3). We must add to this statement the fact that we couldn t identify any case of polyploidy in all the studied specimens. Taking into account the arm ratio (q/p), the difference between arms (q - p), the centromeric index and the relative length we could state that Inula ensifolia L. presents two types of chromosomes: median (m), the pairs of homologues I - VII (having the ratio between arms from 1.03 and 1.53) and one submedian pair of homologues VIII (arm ratio 1.77) (fig. 3). Figure 2: Metaphase Inula ensifolia L. (2n=16) As depicted in table 1, as well as observed on the figures 2 and 3, the total length of chromosomes is ranging between (pair I) and (pair VIII). Knowing the generally accepted fact that the chromosomes length is not the most important criterion for the identification of the pairs of homologues, we were permanently aware that large chromosomes reach their maximum of condensing in late metaphase stages, while small Figure 3: Karyotype - Inula ensifolia L. (2n=16) 194 chromosomes start the contraction process much early. For Inula ensifolia L. (2n=16) the arm ratio (q/p) ranges between 1.03 (pair III) and 1.77 (pair VIII) and the centromeric index ranges between (pair III) and (pair VIII). The haploid set length (HSL) was and the relative length varied between the values: 9.52 (last pair) and (first pair) (table 1). In discordance with the research of Kamari et al., 2008, we could not identify any satellites or

4 secondary constrictions using only classical cytogenetic techniques, but one must be aware that the studies of the Greek researcher were performed on a Mediterranean triploid variant. Because we identified only two types of chromosomes and keeping in mind Stebbins classification, we can also state the fact that the karyotype of Inula hirta L. shows a high degree of symmetry, proving that during its evolution didn t occur important structural rearrangements. We must add to this statement the fact that we couldn t identify any case of polyploidy in all the studies specimens. Based on the average of all our measurements we are proposing an idiogram for the species Inula ensifolia L. (2n = 16) (fig. 4). Chromosome pair Chromosome type Figure 4: Proposed idiogram for Inula ensifolia L. (2n = 16) Total length Long arm Short arm Table 1. Metric traits of Inula ensifolia L. chromosomes Arm ratio (q/p) Difference between arms (q - p) Centromeric Index Relative length I m II m III m IV m V m VI m VII m VIII sm LSH Conclusions We have studied the chromosomes morphology for two species of Inula: Inula ensifolia L., and Inula helenium L. For I. ensifolia (2n = 16), the karyotype is represented by 7 pairs of median (m) and a pair of submedian chromosomes (sm). With I. helenium, we could establish only the number of diploid chromosomes, 2n=20, 195 and we could observe a submedian pair of chromosomes with satelites. Both species show a symmetrical, unevolved karyotype, based on the lack of structural rearrangements and polyploidy. As we know, this paper is the first report of karyological analysis of these species in Romania and our results are convergent with the data reported by foreign authors.

5 References Afemei, M., Tudose, C., Vochita, G.: Cytogenetic studies regarding two species of Inula spiraeifolia L. and Inula hirta L., An. St. Univ. Iasi, Sect. Genet. Biol. Molec.. XIII (3), 2012 (in press) Fedorov, A.A.: Chromosome Numbers of Flowering Plants. Nauka, Leningrad, Goldblatt, P. & Johnson, D.E.: Index to plant chromosome numbers Monogr. Syst. Bot., Missouri Bot. Gard., 30, 2000 Kuzmanov, B.: Chromosome numbers of Bulgarian angiosperms: An introduction to a chromosome atlas of the Bulgarian flora. Fl. Medit. 3, , Kamari, G., Blanché, C., Siljak Yakovlev, S.: Mediterranean chromosome number reports, Flora Medit.,18, , 2008 Kokubugata, G., Kondo, K., Tatarenko, I. V., Kulikov, P.V., Knyasev, M.S., Ryabinina, Z.N.: Cytological studies of 13 Asteraceae species from Russia, Chromosome Science, 6, 67-72, 2002 Levan, A., Fregda, K., Sandberg, A.: Nomenclature for centromeric position on chromosomes. Hereditas, 52(2), , Ma, X.H., Qin, R.L., Xing, W.B., Chromosome observation of twenty species of drug plants in Xingjiang, Acta Bot. Boreal. Occid. Sin., 5: , Morton, J.K.: A cytological study of the Compositae (excluding Hieracium and Taraxacum) of the British Isles, Watsonia, 11, , Rutland, J.P.: The Merton Catalogue: A list of chromosome numbers of British plants, New Phytologist, 40(1): , Tudose, I.: Genetica. Îndrumător de laborator, Lito. Univ., Al. I. Cuza, Iași, 48-52,