Differential Fluorescent Chromosome Banding of Solanum Nigrum L. and Solanum Villosum L
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© 2007 The Japan Mendel Society Cytologia 72(2): 213–219, 2007 Differential Fluorescent Chromosome Banding of Solanum nigrum L. and Solanum villosum L. from Bangladesh Syeda Sharmeen Sultana and Sheikh Shamimul Alam* Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh Received March 5, 2007; accepted April 23, 2007 Summary Two species of the genus Solanum viz. S. nigrum and S. villosum found in Bangladesh were cytogenetically investigated to confirm their taxonomic status. S. nigrum and S. villosum were found to possess 2nϭ24 and 2nϭ48 chromosomes, respectively. The centromeric formula 22mϩ 2sm was found in S. nigrum and 48m in S. villosum. No gradual decrease of chromosomal length was observed in both the species indicated their karyotypes as primitive type. The individual chro- mosomal length ranged from 1.66 to 2.34 mm in S. nigrum and 1.66 to 2.66 mm in S. villosum. The total chromatin length in S. villosum (97.8 mm) was almost double to that of S. nigrum (48.94 mm). The range of relative length of chromosomes was similar in these two species. Solanum nigrum and S. villosum possessed 18 and 17 CMA positive bands, respectively. Most of the CMA-bands were present at the terminal region in both the species. The percentage of CMA banded region in S. villo- sum (35.43) was almost double to that of S. nigrum (18.69). A pair of DAPI positive bands was found on both the end of all the chromosomes in these 2 species. Each band was 0.5 mm in length. The karyotype of S. nigrum studied here indicated that the specimen was not actually S. nigrum rather it has much simillarities with S. americanum. Solanum villosum showed regular bivalent for- mation at metaphase-I and segregation at anaphase-I. The overall karyotypic features suggested S. villosum as an ancient auto-tetraploid of S. americanum which in course of time has started regular meiosis. Key words Fluorescent banding, Karyotype, Solanum nigrum, Solanum villosum. The genus Solanum L. belongs to Solanaceae consisting of 1400 species (Cronquist 1981). Members of this genus have wide distribution and several ethno-botanic importances (Grubben and Denton 2004). Solanum nigrum is a common species found in different parts of the world. This is predomi- nantly grown throughout India, Pakistan and Sri Lanka. It is commonly distributed in all the tem- perate and tropical parts of the world (Khan and Mia 2002). Different forms in respect of the colour and size of berry are found in this species. These are orange, orange-brown, reddish and black. Black form is of 2 types, one with bigger and other smaller sized berry. Initially taxonomists put all these forms in S. nigrum complex (Professor Abul Hassan, Department of Botany, University of Dhaka, Personal communication). However, Schilling and Anderson (1990) recognized these forms as distinct species. They considered the orange, orange-brown or yellow-radish fruited form as S. villosum having four sets of genomes i.e. tetraploid. According to them the relatively big black fruited (1.8–2.2 mm) form is S. nigrum, a hexaploid whereas the smaller black fruited (1.2–1.6 mm) one is a diploid called S. americanum. In Bangladesh, 2 forms of Solanum have so far been reported. One is black and the other or- ange fruited. The black fruited form is widely distributed from Comilla to Chittagong districts. In this area, orange fruited forms are not found or very rare. On the other hand, orange fruited forms * Corresponding author, e-mail: [email protected] 214 Syeda Sharmeen Sultana and Sheikh Shamimul Alam Cytologia 72(2) are widespread in Dhaka district. In spite of the berry colour and size, several taxonomists erro- neously grouped these two forms (black and orange fruited) in S. nigrum (Professor Abul Hassan, Department of Botany, University of Dhaka, Personal communication). Therefore, a confusion regarding the taxonomic rank of the different forms of S. nigrum still exists. With the help of traditional taxonomy it is not possible to overcome this problem. It is now clear that where morphological characters are meager, cytological investigation would help solving the taxonomic problem. An authentic cytogenetical analysis will prove its work to solve the said problem in S. nigrum. Karyotype analysis would be an important aspect to study the taxa of interest. Karyotype is a stable character and specific for each specimen. Sometimes traditional karyotype analysis is not enough to provide sufficient comparative data specially when different taxa possess similar chromo- some numbers and centromeric formula. Even, the consideration of chromosome length, arm ratio, position and number of secondary constrictions are not always sufficient to differentiate individual chromosome. Moreover, minute change in the genome does not help to detect with conventional karyotype analysis. In such a situation, some modern methods may be used. Staining with fluo- rochromes (CMA and DAPI) is one of such methods. Schweizer (1976) for the first time initiated this technique. CMA bind with GC-rich repetitive sequences of the genome and gives characteristic yellow colour bands. On the other hand, DAPI bind with AT-rich repetitive sequences fluorescing characteristic blue bands. With the help of fluorescent staining it was possible to solve the taxonomic problem in several species of Crinum (Alam et al. 1998, Ahmed et al. 2004), Ampelygonum chinense (L.) Lindley and A. salarkhanii Hassan (Alam et al. 2000), Colocasia esculentum and Xanthosoma violaceum (Deen and Alam 2002, Alam and Deen 2002) and different forms of Lasia (Sultana et al. 2006). Therefore, the karyotype analysis with both conventional and fluorescent staining might be helpful to distinguish the different Solanum species with each other. In the present study, 2 species of Solanum viz. S. nigrum (black fruited) and S. villosum (orange fruited) were investigated cytolog- ically with the following objectives: (i)To compare the karyotypes of two species after staining with orcein, CMA and DAPI, (ii) To ascertain the taxonomic status of the black fruited species found in Bangladesh, (iii) Elucidate the nature of genomes present in S. villosum. Materials and methods The following 2 species of Solanum viz Solanum nigrum and Solanum villosum were studied in this investigation. Solanum nigrum L. is a semi erect plant with mean plant height of 40 cm. The leaves are long, thin, not curly, green in colour. Flowers are white with light green stem. Fruits are black in colour. Solanum villosum L. is also a semi erect plant with mean plant height of 30 cm. The leaves are wide, thin, curly and green in colour. Flowers are white with reddish orange fruits. Solanum nigrum L. was collected from Chittagong district whereas S. villosum L. from Dhaka dis- trict. These specimens were maintained in the Botanic garden, Department of Botany, University of Dhaka, Bangladesh. Healthy roots were collected and pretreated with 2 mM 8-hydroxyquinoline for 1 h at room temperature followed by 15 min fixation in 45% acetic acid at 4°C. These were then hydrolysed in a mixture of 1 N HCl and 45% acetic acid (2 : 1) at 60°C for 7 s. The root tips were stained and squashed in 1% acetoorcein. For fluorescent banding, Alam and Kondo’s (1995) method was used with slight modification. After hydrolysing and dissecting, the materials were squashed with 45% acetic acid. The cover glasses were removed quickly on dry ice and allowed to air dry for at least 48 h before study. The air-dried slides were first pre-incubated in McIlvaine’s buffer (pH 7.0) for 30 min followed by Distamycin A (0.1 mg/ml) treatment for 10 min. The slides were rinsed mildly 2007 Fluorescent banding in Solanum spp. 215 in McIlvaine’s buffer supplemented with MgSO4 (5 mM) for 15 min. One drop of CMA (0.1 mg/ml) was added to the materials for 15 min and rinsed with McIlvaine’s buffer with Mg2ϩ for 10 min. Slides were mounted in 50% glycerol and kept at 4°C for overnight before observation. These were observed under Nikon (UFX-IIA) fluorescent microscope with Blue Violet (BV) filter cassette. For direct DAPI-staining, after 48 h of air drying, the slide was first pre-incubated in Mcll- vaine’s buffer (pH 7.0) for 20 min in a humid chamber. The slides were treated in actinomycin D (0.25 mg/ml) for 10 min. The slides were immersed in DAPI solution (0.01 mg/ml) for 20 min and mounted with 50% glycerol. These were observed under Nikon (UFX-IIA) fluorescent microscope with Ultra Violet (UV) filter cassette. For studying meiosis, the young unopened buds of S. villosum were collected from the Botanic Garden, University of Dhaka at 9 a.m. and kept in a watch glass with distilled water. The bud was opened by a pair of forceps. Two–three anthers were taken on a clean slide. A drop of carmine was added to it and kept for 1 min. The anthers were gently tapped with a plastic taper. The debris were discarded. A clean cover slip was placed carefully on the materials. The slide was wrapped with a filter paper and very gently pressed by thumbs. A drop of carmine was added to the corner of the cover slip and observed under microscope. For syudying pollen viability, mature flowers with yellow anthers of Solanum villosum were collected from the Botanic garden, University of Dhaka. One drop of carmine was placed on a clean slide. The mature anthers were touched to the carmine dye in such a way so that the pollen grains came in contact with carmine. A clean cover slip was placed on it and observed under microscope. Results and discussion Two species of Solanum found in Bangladesh viz.