2019 Volume 84 Issue 1 Pages 63-67
Karyotypes of three Piper species viz. P. betle L., P. longum L., and P. retrofractum Vahl were studied with a conventional staining and a fluorescent banding. P. betle, P. longum, and P. retrofractum possessed 2n=68, 2n=48 and 2n=72 chromosomes in root tip cells, respectively. Chromosome number of P. retrofractum (2n=6x=72) is the first report. The three species of Piper showed distinct chromomycin A3 (CMA)- and 4′,6-diamidino-2-phenylindole (DAPI)-band patterns based on the number, location, and size of CMA- and DAPI-bands. The several chromosomes fluoresced overall by CMA or DAPI and these fluorescent bands will be a good marker for chromosome study. The three species were different in respect of conventional karyotype and fluorescent banding pattern. In three Piper species, the comparative karyotype analysis of fluorescent banded chromosomes might suggest that chromosome diversity occurs in aneuploidy and polyploidy.
Piperaceae is a large pantropical family with approximately 3000 species under 8–13 genera. Among these genera, Piper L. is the largest and most important genus consisting of about 1200 species (Burger 1972, Samuel 1987, Kupicha 1993, Mathew et al. 1999). The genus has a wide distribution in tropical and subtropical regions concentrated mainly in South and Central America and South Asia (Pradeepkumar et al. 2003). In Bangladesh, Piper is represented by 10 species viz. P. attenuatum Buch.-Ham. ex Wall, P. betle L., P. hamiltonii C. DC., P. longum L., P. nigrum L., P. peepuloides Roxb., P. retrofractum Vahl, P. rhytidocarpum Hook. f., P. sylvestre Lamk. and P. sylvaticum Roxb. (Ahmed et al. 2009).
Out of them P. betle L. commonly known as “Betel” or “Pan” native to Malaysia, is widely cultivated in tropical regions of the world. The leaves of P. betle are used as carminative, astringent, stimulant, antiseptic, in a headache and cough of children (Tin 2011) and roots are well-known for treatment of malaria and asthma (Ghani 2003, Dwivedi and Tripathi 2014). P. longum or “Pipul” is widely cultivated in South East Asia (Ahmed et al. 2009). The fruit is well-known medicine for diseases of the respiratory tract, muscular pains, inflammation and as a general tonic. P. retrofractum is known as “Choi” cultivated in Thailand, India, China, Malaysia, and the Philippines. The roots contain piperine, piperlongumine or piplartine and dihydro-stigmasterol (Manoj et al. 2004). The fruit has a stimulant, carminative, anthelmintic and expectorant properties which are used in asthma, bronchitis, and consumption (Ghani 2003).
Due to their extensive medicinal properties, the plants of three species have long been using as medicine by local people. Though these plants are least concerned depending on threats, P. longum and P. retrofractum have restricted distribution. In this situation, analysis of genetic diversity becomes essential for the development of conservation, management and multiplication strategies for the available Piper species in Bangladesh. In order to elucidate genetic diversity and proper conservation, authentic characterization of Piper species are required.
Previous researches reported on chromosome numbers and conventional karyotypes of about 16 Piper species (Darlington and Janakiammal 1945, Sharma and Bhattacharyya 1959, Dasgupta and Datta 1976, Samuel 1987, Jose and Sharma 1985, Samuel and Morawetz 1989, Mathew et al. 1999, Vossen and Wessel 2000). Paucity of detail cytological data, as well as the conflicting chromosome numbers reported in the genus so far, may be largely due to the extremely small size but the high number of chromosomes, the inter-clonal variation of chromosome numbers and dense cytoplasmic contents which make difficult in the cytogenetical analysis. The intraspecific variation of somatic chromosome number from previous reports in different Piper species indicated the presence of multiple basic chromosome numbers which is originated through different types of numerical and structural variations of chromosomes (Mathew 1958, Sharma and Bhattacharyya 1959, Smith 1966, Jose and Sharma 1985). The parent having different basic chromosome numbers can lead the breeding program unsuccessful (Huq et al. 2007, Dash et al. 2017). Therefore, in the breeding program, it is essential to gather information regarding genetic makeup of Piper species.
Though the genus provides matters of considerable cytological interest, no cytological information is available for the Piper species growing in Bangladesh. Karyotype of plant species provides authentic information which is useful in systematic, evolution and conservation of plants such as Phyllanthus niruri, P. urinaria and Cucurbita maxima with small chromosomes like Piper (Bennett and Leitch 2005, Skornickova et al. 2007, Guerra 2008). Karyotype analysis with differential chromosome banding is a useful tool for obtaining genomic information about plants. Out of several banding techniques a fluorescent banding with DNA-base specific fluorochromes such as CMA and DAPI is a reliable method for karyotype study (Schweizer 1976, Kondo and Hizume 1982, Alam and Kondo 1995, Jessy et al. 2005, Akhter and Alam 2005, Islam and Alam 2011, Sultana et al. 2011).
In the present study, a full strength cytogenetical analysis was carried out for the first time to determine karyotypic diversity among three Piper species viz. P. betle, P. longum and P. retrofractum with the following aims: to make a full strength orcein-stained karyotype for each species and to compare the fluorescent banding patterns after staining with fluorochromes of CMA and DAPI.
Piper longum L. was collected from Bangladesh Agricultural Research Institute (BARI) and P. betle L. and P. retrofractum L. from the Botanical Garden, Department of Botany, University of Dhaka. Ten individuals of each species were investigated and maintained in the Botanical garden, Department of Botany, University of Dhaka, Bangladesh for further examination.
Root tips (RTs) were collected and pretreated with 2 mM 8-hydroxyquinoline for 1 h at 20–25°C followed by 15 min fixation in 45% acetic acid at 4°C. The pretreated RTs were hydrolyzed for 2–3 min depending on the thickness of root at 65°C in a mixture of 1 M HCl and 45% acetic acid (2 : 1). The RTs were stained and squashed in 1% aceto–orcein. For CMA- and DAPI-banding, a method of Alam and Kondo (1995) was used with a slight modification. After hydrolysis, the materials were dissected and squashed in 45% acetic acid. The coverslips were removed by a dry ice method and the slides were allowed to air dry for at least 24 h before staining. The slides were pre-incubated in Mcllvaine 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 5 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 fluorescence microscope (Eclipse 50i, Nikon) with a blue-violet (BV) filter cassette. For DAPI-staining, after 5–6 h of air drying, the slides were incubated in the buffer (pH 7.0) for 25 min and treated in 0.25 mg mL−1 actinomycin D for 10 min in a humid chamber. The slides were immersed in 0.01 mg mL−1 DAPI solution for 3 h and then rinsed with the buffer for 15 min. Finally, the slides were mounted with 50% glycerol. These were observed under the fluorescence microscope with an ultraviolet (UV) filter cassette.
After conventional orcein staining, the three species showed different somatic chromosome counts such as 2n=68 in P. betle, 2n=48 in P. longum and 2n=72 in P. retrofractum (Figs. 1–3, Table 1).
| Species | 2n | The range of chromosome length (µm) | Total chromosome length (µm) | Mean length of chromosomes (µm) | No. of CMA-bands | Area of CMA-bands in the complement (%) | No. of DAPI-bands | Area of DAPI-bands in the complement (%) |
|---|---|---|---|---|---|---|---|---|
| P. betle L. | 68 | 1.52–3.15 | 148.76 | 2.18 | 5 | 7.18 | 6 | 8.36 |
| P. longum L. | 48 | 1.53–2.84 | 103.28 | 2.15 | 2 | 3.16 | — | — |
| P. retrofractum Vahl | 72 | 1.70–2.83 | 161.71 | 2.24 | 8 | 12.26 | — | — |
The diploid chromosome number of 2n=68 for P. betle was determined in 10 individuals in the present investigation supporting a previous report (Vossen and Wessel 2000). Several different chromosome numbers were reported previously viz. 2n=26 (Vossen and Wessel 2000), 2n=32 (Darlington and Janakiammal 1945, Vossen and Wessel 2000), 2n=42 (Jose and Sharma 1985, Vossen and Wessel 2000), 2n=52 (Samuel and Morawetz 1989, Vossen and Wessel 2000), 2n=58 (Jose and Sharma 1985, Vossen and Wessel 2000), 2n=64 (Sharma and Bhattacharyya 1959, Dasgupta and Datta 1976, Vossen and Wessel 2000), 2n=65 (Samuel and Morawetz 1989), 2n=78 (Jose and Sharma 1985, Mathew 1958, Okada 1986, Vossen and Wessel 2000). The presence of several chromosome numbers in P. betle made a confusion regarding the basic chromosome number. To reveal the intraspecific variation of chromosome numbers should need to investigate in many plants of other localities.
P. longum was found to possess 2n=48 chromosomes in the present study (Figs. 2, 10, Table 1). Same chromosome number was reported by Sharma and Bhattacharyya (1959). In addition, different chromosome number such as 2n=24 (Tjio 1948), 2n=60 (Bai and Subramanian 1985) and 2n=96 (Sharma and Bhattacharyya 1959) were also reported. If the basic chromosome number of this species is considered as x=12, then the plants with 2n=4x=48 observed in this study could be regarded as tetraploid and the species with 2n=2x=24 (Tjio 1948, Sharma and Bhattacharyya 1959), 2n=5x=60 (Bai and Subramanian 1985) and 2n=8x=96 (Sharma and Bhattacharyya 1959) could be regarded as diploid, pentaploid and octaploid, respectively.
In P. retrofractum, 2n=72 chromosomes were observed (Fig. 3, Table 1). Different chromosome numbers were reported previously as 2n=24 (Darlington and Janakiammal 1945, Kumar and Subramanian 1986) and 2n=26 (Samuel and Morawetz 1989). If the basic chromosome number of this species is considered as x=12, then specimen with 2n=72 observed in this study could be regarded as hexaploid. However, 2n=26 (Samuel and Morawetz 1989) might be evolved by the aneuploid change.
In the three species, the range of chromosome length was the almost negligible i.e., difference between small and large chromosomes was about 1.5 µm. In this investigation, the total length of chromosome complement was recorded as 148.76 µm for P. betle (2n=68), 103.28 µm for P. longum (2n=48) and 161.71 µm for P. retrofractum (2n=72) which corresponds with their chromosome number. The average length of the individual chromosome was more or less similar i.e., 2.18 µm in P. betle, 2.15 µm in P. longum and 2.24 µm in P. retrofractum (Table 1).
Fluorescent banding patternFluorescent banding patterns of the three Piper species differed sharply regarding the number and intensities of CMA- and DAPI-bands (Figs. 4–9, 13–18, Table 1). The number of CMA- and DAPI-bands varied from species to species. The area percentage of CMA- and DAPI-bands in chromosome complement was different among different species (Table 1). It has seen that each species has distinct CMA- and DAPI-banded karyotype (Table 1).
After CMA-staining, five, two and eight CMA-bands appeared on a chromosome complement of P. betle, P. longum and P. retrofractum, respectively (Figs. 4–6, Table 1). Among these 15 CMA-banded chromosomes in three species, 14 chromosomes were entirely fluoresced with CMA. Similarly, after DAPI-staining, six chromosomes showed DAPI-bands in P. betle of which five chromosomes were entirely fluoresced with DAPI (Fig. 4, Table 1). In these entirely fluoresced chromosomes, GC- and AT-rich segment might distribute on whole chromosomes. These entirely fluoresced chromosomes might be occurred by tandem duplication of GC- and AT-rich repeats (Schweizer 1976, Hiron et al. 2006, Mahbub et al. 2007, Sultana and Alam 2007, Khatun and Alam 2010). For molecular cytogenetic analysis, isolation of the GC-rich and AT-rich repeats contributing fluorescent bands is desired.
Heteromorphicity in respect of banding pattern was observed in three Piper species after CMA- and DAPI-staining. One chromosome in P. betle was entirely fluoresced with CMA while its homologue member did not show any band (Fig. 13). Similarly, in P. longum, one chromosome and the long arm of another chromosome were entirely fluoresced with CMA whereas no bands were observed in their homologue (Figs. 5, 14). Heteromorphic in respect of banding pattern was also observed after DAPI-banding. A centromeric DAPI-band in one chromosome and entirely DAPI-fluoresced two chromosomes were observed in P. betle (Figs. 7, 16, Table 1). This heterogeneity suggests that the Piper species investigated have intraspecific variations in each species. The standard fluorescent banding karyotypes will be revealed by fluorescent banding analysis in many individuals in each species.
This research was partly supported by a grant from the Ministry of Science and Technology, People’s Republic of Bangladesh.
