Karyomorphological Studies of Two Species of Salvadora (Salvadoraceae) from Western India

American International Journal of Available online at http://www.iasir.net Research in Formal, Applied & Natural Sciences ISSN (Print): 2328-3777, ISSN (Online): 2328-3785, ISSN (CD-ROM): 2328-3793

AIJRFANS is a refereed, indexed, peer-reviewed, multidisciplinary and open access journal published by International Association of Scientific Innovation and Research (IASIR), USA (An Association Unifying the Sciences, Engineering, and Applied Research)

SUSHILA SAINI1, JAYA PARKASH YADAV2 1Assistant Professor, Department of Botany, J.V.M.G.R.R. College, Ch. Dadri, Bhiwani, Haryana, India 2 Professor, Department of Genetics M.D. University, Rohtak, Haryana, India

Abstract: The present research was conducted for karyomorphological investigation on two lesser known but ethanomedicinally and ethanopharmacologically important species of genus Salvadora. For this study, 0.020% colchicine and leucobasic fuchsin were used as pretreatment solution and for staining. Chromosome characteristics including long arm, short arm, chromosome lengths, total length of chromosome set, arm ratio index and centromeric index were measured. The studies showed that the somatic chromosome number in the genus Salvadora was 2n=24. The karyotype formula of S. oleoides Decne. was found to be K (2n) = 24= 3m+ 9sm while that of S. persica L. was K (2n) = 24= 8nm+3nsm+1sm. The chromosomes of both the taxa were medium in length ranging from 2.13 to 4 constriction was observed in any of these species. Symmetry of S. oleoides chromosome was found to be 2A while of S. persica was 2B. This finding may provide useful information for Salvadora evolutionary, genetic and breeding studies.

Keywords: Salvadora oleoides, Salvadora persica, Chromosome, Karyotype, Chromosome symmetry.

I. Introduction

Salvadora is a small genus of evergreen trees or shrubs distributed in tropical Africa and Asia extending up to Mascarene Island and China [10]. Two species of Salvadora found in India are – Salvadora oleoides Dence. (metha jal) and Salvadora persica L. (khara jal). Both the species are highly salt tolerant and extremely well adapted to arid conditions [11] and they contains a number of constituents like terpenoids, fixed oils, steroids, alkaloids, flavanoids, saponins, tannins. These species have a wide spread usage for human medicine and personal hygiene and almost all parts have been found to be pharmaceutically important [9]. Some useful activities reported from various parts of the plants and from the isolated chemicals are- antiulcer, hypolipidemic, hypoglycemic and anticonvulsant activities [13, 16, 8, 12]. Due to the presence of benzyl isothiocynate S. persica is widely used as an oral hygiene tool in different part of the world under the name of Miswak. Scientific evaluation of use of miswak revealed that it is at least as effective as tooth brushing for reducing plaque and gingivitis and that the antimicrobial effect of S. persica is beneficial for prevention of periodontal disease [6]. These species with their immense potential in ethanomedicine are threatened with extinction due to decline in the number of seeds per plant and reduced seed viability. Cytological techniques determine the chromosome constitution of an organism and facilitate recognition of the individual chromosomes. In plant taxonomy, breeding, and genetic studies, information about chromosome karyotype can be useful in species identification and analysis of hybrid populations [14, 15, 20, 7]. In case of Salvadora species chromosome counts have been reported from time to time by various cytologists but detailed description and visual presentation of karyotype of these species have not been published. The data on chromosome number and karyotypic analysis are prerequisites to the overall understanding of the genome. Therefore, an attempt has been made to analyze the detailed karyotypes of S. oleoides and S. persica.

II. Materials and methods The experiment was conducted in the laboratory of the Department of Genetics, MD University, Rohtak (Haryana) India. Seeds samples of both the species were collected from the different regions of Western India. The plant material was authenticated by FRI Dehradun that has allotted accession no. 14/153533 and 10786 for S. oleoides and S. persica respectively. Seed germination was conducted in BOD incubator held at 250C in petriplates lined with moistened Whatman filter paper. For karyotype analysis root tips were pretreated with 0.020% colchicine for 3 hour at 250C and then fixed in freshly prepared Carnoy’s fixative (1 glacial acetic acid: 3 chloroform: 6 ethanol). The root tips that were not required immediately for slide preparation were stored in 70% ethanol in a refrigerator. The root tips were hydrolyzed with 1N HCl at 600C for 8 min. They were stained in 0.5% leucobasic fuchsin for 10 min at 250C. After staining meristematic region with 1mm of length was excised and macerated in acetocarmine for 5 min. karyotype analysis was based on at least seven high quality metaphase cell plates. Chromosomes were measured for long arm, short arm, total length of chromosome set, arm ratio and centromeric index. Chromosome nomenclature and classification was made as per the scheme proposed by Abraham and Prasad [19]. The symmetry of karyotype was classified according to Stebbins [5]. Along with this Total Form percentage (TF %) [18] was also calculated. Total sum of length of short arm of chromosomes TF % = × 100 Total sum of total length of all the chromosomes III. Results S. oleoides: The chromosome number of this species is 2n=24 (figure 1(a)). Karyotypic characters of the mitotic chromosomes are shown in table 1. The karyotype consists of 12 chromosome pairs with 3 pairs of median (m) and 9 pairs of sub median (sm) chromosomes (figure 2(c)). The karyotype formula was found to be K (2n) = 24= 3m+ 9sm. The length of chromosome in this taxon varied from 2.13m to 4.20m. The longest arm is 2.14m and the shortest arm is 0.80m. The total length of haploid complement of S. oleoides chromosome is 37.45m. No secondary constriction was observed in any of these chromosomes. TF % value of S. oleoides chromosome was found to be 41.37. Following the classification of Stebbins the ratio of largest to smallest chromosome was less than 2:1 and arm ratio of less than half chromosome (33%) was more than 2:1. Thus the degree of symmetry in S. oleoides was resolved in to 2A. S. persica: The chromosome number of S. persica is 2n=24 (figure 1(b)). Karyotypic characters of the mitotic chromosomes are shown in table 2. The karyotype consist of 12 chromosome pairs with 8 pairs of nearly median (nm), 3 pairs of nearly sub median (nsm) and one pair of sub median (sm) chromosome (figure 2(d)). The karyotype formula was found to be K (2n) = 24= 8nm+3nsm+1sm. The length of chromosome in this taxon ranged from 2.95m to 6.09m. The longest arm is 4.08m and the shortest arm is 0.91m. The total length of haploid complement of S. persica chromosome is 48.98m. Secondary constriction was not observed in any of these chromosomes. TF % value of S. persica chromosome was found to be 40.11. In case of S. persica the ratio of largest to smallest chromosome was more than 2:1 and arm ratio of less than half chromosome (33%) exceeds 2:1. Thus the degree of symmetry in S. persica was resolved in to 2B. IV. Discussion The diploid chromosome number in both Salvadora species was determined to be 2n = 24. In case of S. persica the chromosome count correlates well with the previous findings of Koul and Singh [1]. On the basis of meiotic analysis they observed chromosome number to be 2n = 24. Subramanian [3] also reported diploid chromosome number in S. persica, 2n = 24 and the total chromatin length was 23.6m. The chromosomes were of medium and short size; ranging in length from 0.8m to 1.2m. The karyotype formula observed was K (2n) = 24 = L6 + M10 + O8. However, some deviant chromosome number likes n = 13 were also reported for S. persica by Sindhu [17] and Sarkar et al. [2]. Baquar and Hussain [4] reported the diploid chromosome number to be 2n = 24 in case of S. oleoides. Thus the present data coincides with the available reports, confirms that Salvadora is a monobasic genus with n=12. Karyotype of both the species shows distinct differences: S. persica chromosomes were longer than S. oleoides in size. The total chromatin length ranged from 2.15m to 4.20 m in case of S. oleoides; while in case of S. persica it ranged from 2.95m to 6.09 m. In case of S. oleoides only median and nearly sub-median chromosomes were present. However S. persica had three chromosome types- nearly median, nearly sub-median

AIJRFANS 13-157; © 2013, AIJRFANS All Rights Reserved Page 15 S. Saini et al., American International Journal of Research in Formal, Applied & Natural Sciences, 2(1), March-May, 2013, pp. 14-18 and sub-median. No nucleolar organizer was observed in the form of secondary constriction in any of these species. Since majority of the chromosomes were of median and sub-median types, karyotypes of both taxa studied were found to be of symmetrical type. TF% value of S. oleoides chromosomes (41.37) was higher than S. persica (40.11), thus its karyotype was more symmetrical than that of S. persica. Within species karyotypic variation was not observed in this study. Incidence of polyploidy / aneuploidy or numerical variation was not reported in any of the Salvadora species. However a clear understanding of genome organization and species differentiation can only be achieved by studying chromosomes using modern techniques of molecular cytogenetics such as in situ hybridization (FISH) and Fluorescence Assisted Chromosome Sorting (FACS) etc.

10 µm (a) (b)

Figure 1: (a) Somatic metaphase chromosome of S. oleoides (b) Somatic metaphase chromosome of S. persica (scale bar equal 10 µm)

(c)

5µm

(d)

Figure 2: (c) Karyotype of metaphase chromosome of S. oleoides (d) Karyotype of metaphase chromosome of S. persica. (scale bars equal 5 µm)

Table 1: Karyotype details of somatic metaphase chromosomes of S. oleoides. Chromosome Long arm Short arm Total Arm ratio Centromeric Chromosome pairs (l) in m (s) in m length (c) in Index nomenclature* m s/l l/s 100s/c 100l/c (R1) (R2) (I1) (I2) 1 2.100.02 2.100.02 4.200.04 1 1 50 50 m 2 2.060.03 2.060.01 4.120.03 1 1 50 50 m 3 2.000.02 2.000.03 4.000.04 1 1 50 50 m 4 2.130.03 1.130.02 3.260.05 0.53 1.88 34.66 65.33 nsm (-) 5 2.140.02 1.010.03 3.150.04 0.47 2.12 32.06 67.93 nsm(-) 6 2.100.01 0.990.03 3.090.04 0.47 2.12 32.03 67.96 nsm (-) 7 2.090.01 0.910.02 3.000.03 0.43 2.29 30.33 69.67 nsm (-) 8 1.970.02 0.920.03 2.890.05 0.47 2.14 31.83 68.17 nsm(-) 9 1.800.02 0.940.03 2.740.05 0.52 1.91 34.30 65.69 nsm (-) 10 1.650.03 0.900.03 2.550.06 0.54 1.83 35.29 64.70 nsm (-) 11 1.450.03 0.870.02 2.320.04 0.60 1.67 37.5 62.5 nsm (-) 12 1.330.01 0.800.02 2.130.03 0.60 1.66 37.55 62.44 nsm (-) *m= median, nsm (-) = nearly submedian.

Table 2: Karyotype details of somatic metaphase chromosomes of S. persica. Chromosome Long arm Short arm Total Arm ratio Centromeric Index Chromosome pairs (l) in m (s) in m length (c) nomenclature* in m s/l l/s 100s/c 100l/c (R1) (R2) (I1) (I2) 1 4.080.01 2.010.02 6.090.03 0.49 2.03 33.00 66.99 nsm (-)

2 3.160.02 2.010.01 5.170.02 0.64 1.57 38.88 61.12 nm 3 2.280.01 2.170.02 4.450.03 0.95 1.05 48.76 51.23 nm 4 2.200.03 2.130.02 4.330.04 0.97 1.03 49.19 50.80 nm 5 2.090.01 2.070.01 4.160.02 0.99 1.01 49.75 50.24 nm 6 2.090.01 2.000.02 4.090.03 0.96 1.04 48.89 51.10 nm 7 3.000.02 1.000.01 4.000.02 0.64 3.00 25.00 75.00 sm 8 2.870.01 0.910.03 3.780.04 0.33 3.15 24.07 74.16 nsm (-) 9 2.500.02 1.080.02 3.580.03 0.43 2.31 30.17 69.83 nsm (-) 10 1.880.02 1.380.03 3.260.04 0.73 1.36 42.33 57.66 nm 11 1.660.03 1.460.02 3.120.05 0.88 1.14 46.79 53.20 nm 12 1.520.02 1.430.02 2.950.04 0.94 1.06 48.47 51.52 nm * nm = nearly median, nsm (-) = nearly submedian, sm = sub median

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