Karyological investigation of Calotropis gigantea (L.) W. T. Aiton and Centella asiatica (L.) Urban: Two important medicinal plants of Bangladesh

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Highlights

  • Two medicinal plants, Calotropis gigantea (L.) W. T. Aiton and Centella asiatica (L.) Urban were analyzed cytogenetically with orcein-, CMA- and DAPI-staining.

  • 2n = 22 and 2n = 18 chromosomes were found in C. gigantea and C. asiatica, respectively.

  • Number, location and amount of CMA- and DAPI-bands were analyzed.

  • TF%, AsK% and Syi% values for these two species were calculated and both species possessed 1A karyotype.

  • Two species can be characterized with cytogenetical data and enhanced the chromosomal database of medicinal plants of Bangladesh.

Abstract

Calotropis gigantea (L.) W. T. Aiton and Centella asiatica (L.) Urban have been medicinally used throughout Bangladesh. In this study, karyotypic features of these two species were studied with orcein staining and banding with two base specific fluorochromes such as chromomycine A3 (CMA) and 4´, 6 diamidino-2-phenylindole (DAPI) for authentic cytogenetical characterization. 2n = 22 and 2n = 18 somatic chromosomes were found in C. gigantea and C. asiatica, respectively. The total chromosome length of C. gigantea and C. asiatica were 45.35 ± 2.64 μm and 63.63 ± 3.19 μm, respectively. The total form percent (TF%), karyotype asymmetry index (AsK%) and karyotype symmetry index (Syi index) were 47.61, 52.39 and 90.87 in C. gigantea and 39.58, 60.42 and 65.51 in C. asiatica, respectively. Both species were found to possess 1A karyotype. In C. gigantea, 2 CMA- and 4 DAPI-bands were observed whereas C. asiatica had 16 CMA- and 6 DAPI-bands. Therefore, the compiled data generated from chromosome analysis on orcein, CMA- and DAPI-banding will be useful for cytogenetical characterization of these two species.

Introduction

Bangladesh has tremendous wealth of medicinal plants in terms of genetic diversity. Calotropis gigantea (L.) W. T. Aiton and Centella asiatica (L.) Urban are the top listed medicinal plants of Bangladesh used by the local and ethnic people for hundreds of years. Calotropis gigantea belongs to Asclepiadaceae, locally known as Crown Flower, Giant Milkweed or Akand, is widely distributed in China, India, Indonesia, Malaysia, Myanmar, Nepal and Thailand. In Bangladesh, it occurs throughout the country (Ahmed et al., 2008). The whole part of C. gigantea is used for the treatment of various diseases. Root bark of this plant is diaphoretic, emetic, alterative and purgative; useful in dysentery, asthma, elephantiasis, syphilis and heart diseases (Das, 1996; Yusuf et al., 2009). It is effective in scabies and ringworm of the scalp and piles. Leaves of C. gigantea are used as poultice against rheumatism, chest pain due to cold, paralyzed parts and in dropsy. It is also useful in skin diseases, wounds and insect bites (Yusuf et al., 2009).

Centella asiatica of Apiaceae family, is commonly known as Asiatic pennywort, Indian pennywort, Indian water navelwort, Wild violet (in English) and Thankuni (in Bangla), distributed throughout tropical and sub-tropical regions of the world. In Bangladesh, it grows naturally in all parts of the country (Ahmed et al., 2008). Sometimes it is commercially cultivated for enormous medicinal and economic value. Centella asiatica is astringent, tonic, diuretic, laxative, digestive and anti-pyretic; improves appetite, voice and memory; cures dysentery, loose motion, stomach pain, leucoderma, urinary discharges, bronchitis, inflammations, fevers, convulsive disorders, insanity and syphilitic skin diseases (Yusuf et al., 2009; Prakash et al., 2017). It’s efficacy as an internal and external remedy in ulcerations, eczema, leprosy and other coetaneous affections are established. It has also a special influence on the genitourinary tracts (Yusuf et al., 2009).

According to previous records, the studied species C. gigantea represents a mono-basic chromosome number i.e. x = 11 with somatic chromosome number 2n = 22 (Table 1). No other chromosome number has been reported for C. gigantea so far. Almost all the previous research on C. gigantea was confined to chromosome count without karyotyping. On the other hand, C. asiatica possesses different somatic chromosome number such as 2n = 18, 2n = 22, 2n = 33, 2n = 36, 2n = 54 and 2n = 76 according to available literatures. B-chromosomes were also reported in C. asiatica (Raghuvanshi and Joshi, 1968; Joshi and Raghuvanshi, 1970) (Table 1). Rakotondralambo et al. (2013) reported some diploids and tetraploid populations of C. asiatica from Madagascar. In Bangladesh, conventional cytogenetical analysis of C. asiatica was available only with orcein staining (Warasy, 2015). However, less cytogenetical data was available for C. gigantea. As these two species are medicinally important, their genetic information should be investigated. For this purpose, two counter DNA base specific fluorescent banding methods such as chromomycine A3 (CMA) and 4´, 6 diamidino-2-phenylindole (DAPI) along with orcein-staining were used for cytogenetical analysis. CMA binds with GC (Guanine-Cytosine)-rich repetitive sequences of the genome expressing yellow fluorescence. On the other hand, DAPI binds to AT (Adenine-Thymine)-rich repeats giving characteristic blue color (Alam and Kondo, 1995; Dash et al., 2017). Although C. gigantea and C. asiatica are medicinally important, no modern cytogenetical research has so far been initiated to characterize each species in Bangladesh. The characterization with genetic makeup is essential for its proper conservation and management. Therefore, the aim of present research was to characterize C. gigantea and C. asiatica with karyotypic parameters through differential chromosome banding and enhance the chromosomal database of medicinal plants in Bangladesh.

Section snippets

Material and methods

In this study, Calotropis gigantea (L.) W. T. Aiton and Centella asiatica (L.) Urban were used as materials. These plants were maintained in the Botanical Garden, Department of Botany, University of Dhaka. In this investigation, five individuals of Calotropis gigantea and twenty individuals of Centella asiatica were used as materials. At least 50 healthy root tips (RTs) of each species were collected and pretreated with 2 mM 8-hydroxyquinoline for 1 h and 30 min at 20–25 °C followed by 15 min

Calotropis gigantea (L.) W. T. Aiton

In the genus Calotropis, four chromosome numbers were reported earlier for different species such as 2n = 22 in C. gigantea, C. procera, C. nilotica, C. mozambicensis, C. sandersonii; 2n = 26 in C. procera and 2n = 44, 48 in C. woodii (Fedorov, 1974; Kumar and Subramaniam, 1987). Most of the species under the genus Calotropis showed x = 11 basic chromosome number. However, C. procera and C. woodii showed two different chromosome number i.e. 2n = 22, 26 and 2n = 44, 48, respectively.

Conclusions

The studied Calotropis gigantea (L.) W. T. Aiton and Centella asiatica (L.) Urban was diploid with chromosome number of 2n = 22 (x = 11) and 2n = 18 (x = 9), respectively. C. gigantea had symmetric karyotype while C. asiatica were slightly asymmetric. Both species could be categorized in 1A group of karyotype indicating primitive nature in terms of evolutionary point of view. The karyotype profile of these two species with extended karyomorphological features could enrich the chromosomal

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors’ contributions

Dash, C.K. performs the current study, analyzed the data and wrote the manuscript, Rahman, M.S. and Afroz, M. collect the species, analyzed the data, wrote and review the manuscript. Sultana, S.S. is the advisor who designed the experiments, analyzed and interpreted the data and critically reviewed the manuscript. All authors read and approved the final manuscript and have made substantive intellectual contributions to the manuscript.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgments

This research is dedicated by the authors to Late Professor Dr. Sheikh Shamimul Alam for his eminent contribution in Cytogenetics Laboratory, University of Dhaka, Bangladesh.

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