Regular Article
Chromosomal Characterization in Plumbago zeylanica and P. indica from Bangladesh
2019 Volume 84 Issue 2 Pages 153-155
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2019 Volume 84 Issue 2 Pages 153-155
Two species of Plumbago from Bangladesh viz. P. zeylanica L. and P. indica L. were studied cytogenetically for authentic characterization. P. zeylanica and P. indica possessed 2n=28 and 2n=14 somatic chromosomes, respectively. These species differed in respect of total length of chromosomes in the complement, range of chromosome length and number of satellites. The two species showed distinct chromomycin A3 (CMA)- and 4′,6-diamidino-2-phenylindole (DAPI)-banding patterns. P. zeylanica and P. indica showed 11 and four CMA-bands, and six and two DAPI-bands, respectively. Several entirely fluoresced chromosomes were observed in both species after CMA- and DAPI-bandings. The difference in number, location and amount of CMA- and DAPI-bands suggested chromosomal diversification between the two species.
The genus Plumbago, Plumbaginaceae is comprising of about 15–20 species (Chetty et al. 1998). Two species of Plumbago namely P. indica L. and P. zeylanica L. have been reported in Bangladesh. P. zeylanica is found in Dhaka, Jamalpur and Panchagarh districts whereas P. indica has been distributed in Munshigonj district and the Chittagong Hill Tracts. These are mainly perennial herbs or under shrubs, grown in waste places and monsoon forests (Ahmed et al. 2009). P. zeylanica is an important medicinal plant because of its high therapeutic uses from ancient times. P. indica is found to be grown in wild conditions but also cultivated as an ornamental plant. This plant has immense medicinal values. Due to overexploitation and the increasing demand of these two Plumbago species for pharmaceutical uses has caused a serious reduction in their native populations. P. indica is now considered as “Conservation Dependent” and in this case, ex-situ conservation is required. On the other hand, the conservation status of P. zeylanica is rare and restricted. So, both in-situ and ex-situ conservation measures should be undertaken for these two species (Ahmed et al. 2009).
In this situation, genetic characterization should be done for the development of suitable conservation strategies of these species. Most of the research work on Plumbago were pharmacology based. A few researchers had done only 2n chromosome counting for cytogenetical characterization of five Plumbago species viz. P. auriculata Lam., P. europaea L., P. indica L., P. pulchella Boiss. and P. zeylanica L. (Sobti and Singh 1961, Dahlgren 1964, Speta 1974, Sanjappa and Sathyananda 1979, Paiva and Leitao 1987, Khatoon and Ali 1993, Nabis 2007). Therefore, authentic characterization of Plumbago species based on cytogenetical data becomes essential for developing the chromosome database of medicinal plants of Bangladesh. For authentic identification, reliable and stable parameters are required. Conventional orcein staining and fluorescent banding techniques could be regarded as an admirable tool for chromosome study (Hiron et al. 2006, Khatun and Alam 2010, Sultana and Alam 2016).
Thus, in the present investigation, chromosome analysis was carried out by a combination of conventional staining and fluorescent banding using CMA and DAPI to characterize P. zeylanica L. and P. indica L. from Bangladesh.
Two Plumbago species of P. zeylanica L. and P. indica L. were collected in and planted in the Botanical Garden of University of Dhaka. In this investigation, five individuals of each species were used as materials. Healthy root tips (RTs) were collected and pretreated with 2 mM 8-hydroxyquinoline for 45 min at 20–25°C followed by 15 min fixation in 45% acetic acid at 4°C. The RTs were hydrolyzed for 3–4 min depending on a thickness of root at 64°C in a mixture of 1 M HCl and 45% acetic acid (2 : 1). A drop of 1% aceto-orcein was added to the material and kept in an acetic acid chamber for overnight. For CMA- and DAPI-bandings, the method of Alam and Kondo (1995) was used with slight modification. After the hydrolyzing, the dissected meristematic portion of RTs was squashed with 45% acetic acid. The cover glasses were removed quickly on dry ice and allowed to air dry for at least 24 h before the study. The air-dried slides were first incubated in McIlvaine buffer (pH 7.0) for 30 min followed by 0.1 mg mL−1 distamycin A treatment for 10 min. The slides were rinsed mildly in the buffer supplemented with 5 mM MgSO4 for 15 min. One drop of 0.1 mg mL−1 CMA was added to the materials for 4 h in a humid chamber and then rinsed with the buffer with MgSO4 for 10 min. Slides were mounted in 50% glycerol and kept at 4°C overnight before observation. These were observed under a Nikon fluorescent microscope (Eclipse 50i) with a blue-violet filter cassette. For DAPI-banding, after 24 h of air drying, the slides were incubated in the buffer for 30 min and treated in 0.25 mg mL−1 actinomycin D for 10 min in a humid chamber. The slides were immersed in 0.1 mg mL−1 DAPI solution for 4 h and mounted with 50% glycerol. These were observed under the fluorescent microscope with an ultraviolet filter cassette.
P. zeylanica and P. indica possessed somatic chromosome number of 2n=28 and 2n=14, respectively (Figs. 1, 4, Table 1). In P. zeylanica, 2n=28 chromosome number count has supported the previous reports (Dahlgren 1964, Paiva and Leitao 1987, Khatoon and Ali 1993). However, different chromosome numbers for this species were reported such as 2n=16 (Dahlgren 1964, Sanjappa and Sathyananda 1979) and 2n=14 (Dahlgren 1964). If the basic chromosome number of P. zeylanica is considered as x=7, then the species with 2n=14 (Dahlgren 1964) and 2n=28 (Dahlgren 1964, Paiva and Leitao 1987, Khatoon and Ali 1993) could be regarded as diploid and tetraploid, respectively. In contrast, 2n=16 for this species might be a case of aneuploidy and it should be confirmed by more investigation on this species of other localities.
| Species | 2n | Total length of chromosome complement (µm) | Range of chromosome length (µm) | No. of CMA-bands | Amount of CMA-bands (µm) | Length % of CMA-bands | No. of DAPI-bands | Amount of DAPI-bands (µm) | Length % of DAPI-bands | No. of satellites |
|---|---|---|---|---|---|---|---|---|---|---|
| P. zeylanica L. | 28 | 92.81±2.98 | 1.08±0.16–4.51±0.32 | 11 | 25.23±1.88 | 27.18 | 6 | 11.13±1.68 | 11.99 | 4 |
| P. indica L. | 14 | 36.73±2.60 | 2.09±0.14–3.43±0.24 | 4 | 6.74±1.50 | 18.35 | 2 | 5.02±1.83 | 13.67 | 2 |
Chromosome number of 2n=14 was observed in P. indica (Fig. 4, Table 1) and was the same as the previous report (Sobti and Singh 1961). On the other hand, Dahlgren (1964) and Nabis (2007) reported 2n=28 and 2n=12 chromosomes in P. indica, respectively. If the basic chromosome number of P. indica is considered as x=7, then the species with 2n=14 (Sobti and Singh 1961) and 2n=28 (Dahlgren 1964) could be regarded as diploid and tetraploid, respectively. In contrast, 2n=12 (Nabis 2007) for this species need more study. From the previous reports, the Plumbago is not stable in respect of chromosome number and polyploidy occurs in both species.
The total chromosome length was found 92.81±2.98 µm in P. zeylanica while 36.73±2.60 µm in P. indica (Table 1). The difference between the smallest and largest chromosome in P. zeylanica (1.08±0.16–4.51±0.32 µm) was about 3.5 µm and thus showed a gradual decrease in chromosome length. This observation suggested the heterogeneous nature of chromosome length (Fig. 1, Table 1). In chromosome complement of P. indica, the range of chromosome length was 2.09±0.14–3.43±0.24 µm, a difference between the smallest and largest chromosome was about 1.5 µm (Fig. 4, Table 1). These features indicated that P. indica showed homogeneity in chromosomal size.
Two fluorochromes of CMA and DAPI were used for fluorescent banding analysis of two Plumbago species for the first time. A number of CMA- and DAPI-bands were found in the metaphase chromosomes of two species (Figs. 2, 3, 5, 6, Table 1). P. zeylanica and P. indica showed 11 and four CMA-bands, and six and two DAPI-bands, respectively. After CMA-banding five entirely fluoresced chromosomes were observed in P. zeylanica and two entirely CMA-banded chromosomes were also found in P. indica (Figs. 2, 5). In DAPI banding, a pair of entirely fluoresced chromosomes was observed in both species (Figs. 3, 6). The above observation suggested possible tandem duplication of GC- and AT-rich repeats throughout the whole length of respective chromosomes (Hiron et al. 2006, Khatun and Alam 2010, Sultana and Alam 2016). Six and one terminal CMA-bands were found in P. zeylanica and P. indica, respectively. P. indica showed CMA-bands at the middle portion of one chromosome. After DAPI-banding in P. zeylanica, two chromosomes had terminal DAPI-band and another two chromosomes showed DAPI-bands at the middle portion (Fig. 3). In P. zeylanica the total length of CMA- and DAPI-banded segments was 25.23±1.88 µm and 11.13±1.68 µm that covered about 27.18% and 11.99% of total chromatin length, respectively (Table 1). The total length of CMA- and DAPI-banded segments in P. indica was 6.74±1.50 µm and 5.02±1.83 µm occupying about 18.35% and 13.67% of total chromosome length, respectively (Table 1).
Four satellites were observed in P. zeylanica after orcein-staining, CMA- and DAPI-banding (Figs. 1–3, arrow). In orcein-staining and CMA-banding, a pair of satellites was observed in P. indica (Figs. 4, 5, arrow). After DAPI-banding no satellite was found in this species (Fig. 6). The satellites of P. indica might be composed of GC-rich sequences; as a result, those were undetected with DAPI.
In this investigation, these two Plumbago species showed different CMA- and DAPI-banding patterns based on number, location and amount of fluorescent bands. The chromosome analysis by conventional and fluorescent bandings provides preliminary cytogenetical data in two Plumbago species and more investigations on other species under the genus Plumbago with many individuals of several localities is needed to understand the possible chromosomal relationship among species of this genus.
This research was partly supported by a grant from the Ministry of Science and Technology, People’s Republic of Bangladesh.
