© 2006 The Japan Mendel Society Cytologia 71(4): 359–364, 2006 Karyotype Analysis in Three Morphological Forms of Lasia spinosa (L.) Thwaites (Araceae) Sharmin Sultana, Rubaiyath Bin Rahman and Sheikh Shamimul Alam* Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh Received July 24, 2006; accepted September 12, 2006 Summary Three morphological forms based on the leaf character of Lasia spinosa (L.) Thwaites viz. i) sagittate, ii) lamina dissected and iii) a mixed of sagittate and lamina dissected were cytogenet- ically investigated. The sagittate form had 2nϭ27. A minute subtelocentric chromosome was found in the sagittate form. Both lamina dissected and mixed form possessed 2nϭ26 chromosomes. The centromeric formula of sagittate form was 19mϩ7smϩ1st. It was 9mϩ15smϩ2st in the lamina dis- sected form and 14mϩ11smϩ1t in the mixed form. The total length of 2n chromosome complement was 102.23 mm in the sagittate form, 57.78 mm in the lamina dissected form and 74.46 mm in the mixed form. In orcein-staining, one satellite was found in sagittate form, 2 in the lamina dissected form and 5 in the mixed form. In CMA-staining, 2 big CMA-positive satellites were found in the sagittate form. Two small CMA-positive satellites were found in the lamina dissected form. The mixed form had 1 big and 1 small satellite. Two CMA-positive bands were found in sagittate, 5 in lamina dissected and 3 in mixed form. The percentage of GC-rich region was 2.88 in the sagittate, 11.23 in the lamina dissected and 6.79 in the mixed form. The above features indicated that the mixed form might be a natural hybrid between the sagittate and the lamina dissected form. Each form possessed specific karyotype which could be applied to elucidate the taxonomic rank of these 3 forms in Lasia spinosa. Key words CMA banding, Karyotype, Lasia, Araceae. The genus Lasia Lour belongs to the family Araceae. Unlike the other genus of Araceae, Lasia has only 2 species so far been reported (Hosne Ara 2001). These are i) Lasia spinosa (L.) Thwaites and ii) L. concinna Alderw. Lasia spinosa has wide distribution from India (Sikkim, The Himalayas and Assam), Myanmar, Sri Lanka, Singapore, Malaysia and China where as Lasia concinna is con- fined in Indonesia (Hosne Ara 2001, Hay 1988). Although Lasia has only 2 species, Lasia spinosa was found to possess 4 different forms in Bangladesh which mainly based on the morphology of leaves (Hosne Ara 2001). These are i) sagit- tate form, the leaves are sagittate i.e. arrow shape, ii) lamina dissected form, the leaves have several dissection on margin, iii) entire lamina margin form, the leaves have entire margin and iv) a mixed form of sagittate and lamina dissected, this form possesses both sagittate and lamina dissected leaves. Hossain and Sharif (1984) were tried to distinguish taxonomically the different forms of Lasia spinosa. However, they could not find any valid reason of these variations and recognized these as “ecophenic variations”. Identification problems were also found in other members of Araceae. For examples, Coloca- sia esculenta has 3 and Xanthosoma violaceum has 2 forms. Taxonomists faced problems to identi- fy these taxa authentically (Hosne Ara 2000). Latter extensive cytological investigation had been carried out in these taxa. The cytological data indicated sharp differences among the different forms of Colocasia esculenta and Xanthosoma violaceum (Deen and Alam 2002, Alam and Deen 2002). * Corresponding author, e-mail: [email protected] 360 S. Sultana, R. B. Rahman and S. S. Alam Cytologia 71(4) It is well-known that where the taxonomic parameters are insufficient, karyotype analysis often plays an important role in determining the taxonomic status of a taxon. However, when the different taxa possess same chromosome number and almost similar karyotypic features, it is hard to distin- guish between such taxa by conventional karyotype analysis. In such a situation, some modern methods may be used. Staining with fluorochromes (CMA and DAPI) is one of such methods. With the help of fluorescent staining it was possible to characterize different karyotypes. (Schweizer 1976, Alam and Kondo 1995, Alam et al. 1995, Kondo and Hizume 1982, Jessy et al. 2005, Akter and Alam 2005). Therefore the karyotype analysis with both conventional and fluorescent staining might be helpful to distinguish the different forms of Lasia spinosa. In this study, 3 forms of Lasia spinosa viz. i) sagittate form, ii) lamina dissected form and iii) mixed form were investigated cytologically. The aim was to i) compare the karyotypes of 3 forms after staining with orcein and CMA, ii) ascer- tain the status of the mixed form and iii) elucidate whether the different forms of Lasia spinosa could be splited on the basis of differentially stained karyotypes. Materials and methods The 3 forms of Lasia spinosa viz. sagittate, lamina dissected, and mixed were studied in this investigation. These forms were collected from the greater Sylhet and Mymensingh districts of Bangladesh. The plants were grown and maintained in the Botanic garden, Department of Botany, University of Dhaka, Bangladesh. Healthy roots were collected and pretreated with 2 mM 8-hydroxyquinoline for 2.5 h at room temperature followed by 15 min fixation in 45% acetic acid at 4°C. These were then hydrolyzed 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% aceto orcein. For fluorescent banding, Alam and Kondo’s (1995) method was used with slight modification. After hydrolyzing 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 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 30 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. Results and discussion Orcein stained karyotype Although the lamina dissected form and the mixed form were found to possess 2nϭ26 chro- mosomes (Figs. 2, 3), and 2nϭ27 chromosomes were observed in the sagittate form. The extra chromosome in this form was subtelocentric and much smaller than the smallest chromosome (Fig. 1, arrowhead). This chromosome was stable and found in every cell, therefore could not be consid- ered as B-chromosome. Srebniak et al. (2002) found a small chromosome in potato. They have con- sidered this kind of chromosome as mini chromosome. Mini chromosome might be eu- or hetero- chromatic in nature. Sometimes they played vital role in expressing phenotypic characters. Here the function of this mini chromosome was unknown. However, it was a characteristic feature of the karyotype in the sagittate form. The centromeric formula of 19mϩ7smϩ1st was determined in the sagittate form, 9mϩ15smϩ2st in the lamina dissected form and 14mϩ11smϩ1t in the mixed form (Table 1). The 2006 Karyotype Analysis in Lasia 361 Figs. 1–9. Differential staining in three forms of Lasia spinosa. 1) Orcein stained metaphase chromosomes of the sagittate form, 2) Orcein stained metaphase chromosomes of the lamina dissected form, 3) Orcein stained metaphase chromosomes of the mixed form, 4) CMA-stained metaphase chro- mosomes of the sagittate form, 5) CMA-stained metaphase chromosomes of the lamina dissect- ed form, 6) CMA-stained metaphase chromosomes of the mixed form, 7) CMA-stained kary- otype of the sagittate form, 8) CMA-stained karyotype of the lamina dissected form, 9) CMA- stained karyotype of the mixed form. Barϭ10 mm. mixed form showed an intermediate feature in respect of the number of metacentric and sub-meta- centric chromosomes. The 3 forms had metacentric, sub-metacentric, sub-telocentric and telocen- tric chromosomes in their karyotypes. This data revealed that these forms had asymmetric kary- otype. Stebbins (1971) stated that the asymmetric karyotype was advanced character. Thus these 3 forms might be considered as advanced in this regards. 362 S. Sultana, R. B. Rahman and S. S. Alam Cytologia 71(4) Table 1. Comparative orcein and CMA-karyotype analysis in 3 forms of Lasia spinosa (L.) Thwaites Total No. of CMA- GC-rich No. of satellites chromatin Orcein positive bands portion (%) Specimens length karyotypes (including (including Orcein CMA (mm) satellites) satellites) Sagittate form 1 2 102.23 19mϩ7smϩ1st 4 2.88 Lamina dissected form 2 2 57.78 9m ϩ15smϩ2st 7 11.23 Mixed form 5 2 74.46 14mϩ11smϩ1t 5 6.79 mϭmetacentric chromosome, smϭsubmetacentric chromosome, stϭsubtelocentric chromosome, tϭtelocentric chro- mosome. One satellite was found in the sagittate form, 2 in the lamina dissected form and 5 in the mixed form (Figs. 1–3, arrow). The satellite-bearing chromosomes were different in different forms. Therefore, the number and location of satellites were another characteristic feature of the kary- otypes in 3 forms. Heteromorphicity in respect of centromeric type and chromosomal length was recorded in pair XI in the sagittate form, pair I, IV, XIII in the lamina dissected form and pair XIII in the mixed form (Figs. 7–9). The probable reason for this heteromorphicity might be due to deletion from the respective chromosomes. CMA staining Satellites Two big CMA-positive satellites were observed in the sagittate form. These 2 satel- lite-bearing chromosomes were the members of pair V (Figs.