© 2021 The Japan Mendel Society Cytologia 86(1): 41–45 A Report of Karyotype and B-Chromosome in Costus spicatus (Jacq.) Sw. from Bangladesh Susmita Saha, Faria Akbar and Kazi Nahida Begum* Department of Botany, Faculty of Life and Earth Sciences, Jagannath University, Chittaranjan Avenue, Dhaka-1100, Bangladesh Received October 4, 2020; accepted November 4, 2020 Summary Costus spicatus (Jacq.) Sw. is a species of Costaceae (Zingiberaceae) family, which is most familiar as a medicinal plant because of its therapeutic values. A detailed karyological analysis has been accomplished through aceto-orcein staining, chromosome number was reported 2n=18 along with the presence of one B- chromosome, which exhibited with a karyotype formula as 16m+2sm. It would be a new report for C. spicatus. The esteems of various karyomorphological indices regarding the aspect of asymmetry, demonstrated its sym- metry in the karyotype. Therefore, the basic chromosomal information of the current investigation will contribute to deposit chromosomal knowledge and conservation of plant genetic resources of C. spicatus as an invaluable medicinal plant. Keywords Orcein stain, Karyomorphology, Symmetry, B-chromosome, Costus spicatus. Costus is one of the wide-ranging genera of Costaceae serum total cholesterol, LDL-cholesterol, triglyceride, (Zingiberaceae) family consists of about 175 species dis- and when enhancing plasma insulin, HDL-cholesterol tributed to the most part of Neotropical region (Specht and tissue glycogen (Jothivel et al. 2007). et al. 2001, Benny 2004, Specht and Stevenson 2006, As of late, the need for medicinal plants are gradu- Eevera et al. 2010). In this genus, C. spicatus (Jacq.) ally expanded because of their minor or negligible side Sw. is well-known for its medicinal importance as well effects in contrast to manufactured medicines. As a as used as an ornamental plant due to its floral beauty. result, the interest of both domestic and multinational Generally, C. spicatus is known as spiked spiral flag pharmaceutical companies has been expanded into me- ginger or Indian ginger head, found in Southeast Asia. dicinal plants for manufacturing herbal-based medicine In recent periods, it is more profusely found in India and to contribute economically to the global medical indus- Sri Lanka along with Indo-Malayan regions and natural- try as well as the local sector. By reason of increasing ized in some tropical territories (Pawar and Pawar 2014). demands of C. spicatus as a superior medicinal plant and According to Ososki et al. (2002), C. spicatus is sig- delayed vegetative propagation as most of the seeds are nificantly utilized as an anti-diabetic medicinal plant non-viable (Nagendra and Abraham 1981), it is essential throughout the archive of medicinal traditions particu- to take vital steps for the proper conservation of this larly in the Dominican Republic and the United States. medicinal plant. So, the classical cytogenetical analyses Now-a-days, scientific evidence along with traditional are still required because they come up with important evidence furnished that this plant has the potential- evidence regarding numerical and structural features of ity to inverse diabetics and its complexities (Moosmann the chromosome set for karyotype construction in order and Behl 2002). In the Amazon region, C. spicatus is to proper conservation (Akbar et al. 2020, Saha et al. used as a folk and traditional medicinal remedy to cure 2020). pneumonia, skin diseases, asthma, cough, fever, urinary In consequence, the current karyological analysis disease, dysentery and constipation (Quintans Júnior leads to serve cytological knowledge by providing a new et al. 2010). Azhagumadhavan et al. (2019) reported the report on karyotype and B-chromosome with detailed presence of diosgenin, dioscin, saponin, glucosides and karyomorphological analysis on C. spicatus for the first quinine in C. spicatus and they have effective pharma- time from Bangladesh which may help in conservation cological activities such as anti-microbial, anti-choline of genetic resources. esterase, anti-inflammatory, anti-oxidant, antipyretic, anti-hyperglycemic, anti-stress and estrogenic proper- Materials and methods ties. Moreover, C. spicatus has the potentiality to reduce Plant material * Corresponding author, e-mail: [email protected] Five individuals of Costus spicatus (Jacq.) Sw. grow- DOI: 10.1508/cytologia.86.41 ing in the natural population within the wild condition of 42 S. Saha et al. Cytologia 86(1) Fig. 1. Morphology of C. spicatus. (a) plants in natural population, (b) rhizome, scale bar=2.50 cm and (c) compacted cone like spike inflorescence with flower, scale bar=2.85 cm. the Botanical Garden of Department of Botany, Jagan- five analyzed plants. Based on the decreasing order of nath University were collected as analyzed material for chromosome size an idiogram was prepared. According conducting this study and showed in Fig. 1. to the nomenclature of Levan et al. (1964), the karyo- typic formula was determined considering the position Chromosome analysis of the centromere. A divergent range of karyomorpho- Fresh root tips (RTs) with 0.5–1 cm length, were col- logical parameters including symmetry and asymmetry lected from the five plants and initially washed with indices were estimated as the total form percent (TF%) water. Then RTs were pretreated with 2 mM 8-hydroxy- (Huziwara 1962), karyotype asymmetry index (AsK%) quinoline at room temperature (28–30°C) for 50 min. (Arano 1963), the index of karyotype symmetry (Syi%) Afterward, the pre-treated RTs were fixed in Carnoy’s (Greilhuber and Speta 1976), the index of chromosomal fluid (1 glacial acetic acid : 3 ethanol) for 24 h at 4°C. size resemblance (Rec%) (Greilhuber and Speta 1976), For further use, preservation was done in 70% ethanol. intrachromosomal and interchromosomal asymmetry Through a spirit burner, the preserved pre-treated RTs index (A1 and A2) (Zarco 1986), degree of asymmetry of were heated gently with a (3 : 1) mixture of 1% aceto- karyotypes (A) (Watanabe et al. 1999), the asymmetry orcein and 1 M HCl. Lastly, the meristematic portion of index (AI) (Paszko 2006) and Stebbins’s classification RT was squashed gently with a drop of 1% aceto-orcein (Stebbins 1971). under a coverslip. Prepared chromosome slides were ob- served under an Optika microscope and the microscopic Results and discussion photographs were captured with a Euromax camera (CMEX 10, DC 10000C). From the well scattered mitotic metaphases, somatic For somatic chromosome count and karyotype analy- chromosome number, numerical data of karyotype pa- sis three best scattered metaphase of each plant was rameters, idiogram and karyomorphological features of considered, whereas 25 metaphases were observed for C. spicatus were summarized in Fig. 2 and Table 1. confirming the presence of B-chromosome from RTs of In the current investigation, after doing a close inqui- 2021 Karyotype and B-Chromosome Study in Costus spicatus 43 Fig. 2. Orcein-stained mitotic metaphase chromosomes and haploid idiogram of C. spicatus. (a) mitotic metaphase chromo- somes without B-chromosome, (b) mitotic metaphase chromosomes with B-chromosome (indicated by an arrow), (c) haploid idiogram. B indicates B-chromosome. Scale bars=5 µm. Table 1. Numerical data on the karyotype parameters of the haploid chromosome complement of C. spicatus. Chromosome Long arm length±SD Short arm length±SD Total length ±SD (µm) Arm ratio±SD CI±SD CT pair (µm) (µm) I 2.31±0.34 1.60±0.10 3.91±0.25 1.44±0.28 40.92±3.24 m II 2.41±0.05 1.21±0.05 3.62±0.09 1.99±0.07 33.43±1.21 sm III 1.98±0.29 1.38±0.33 3.35±0.10 1.44±0.37 41.04±3.10 m IV 1.73±0.20 1.33±0.17 3.06±0.18 1.31±0.23 43.37±2.77 m V 1.62±0.40 1.20±0.04 2.81±0.02 1.35±0.07 42.53±3.22 m VI 1.48±0.14 1.23±0.12 2.70±0.04 1.20±0.15 45.37±2.51 m VII 1.41±0.09 1.15±0.10 2.56±0.08 1.23±0.09 44.92±2.12 m VIII 1.34±0.05 1.13±0.13 2.46±0.18 1.19±0.15 45.73±2.37 m IX 1.15±0.03 0.98±0.05 2.13±0.07 1.18±0.14 45.88±1.25 m B 0.45±0.12 0.40±0.09 0.85±0.27 1.13±0.11 47.06±0.33 m CI=centromeric index, CT=centromeric type, m=metacentric chromosome, sm=sub-metacentric chromosome, SD=standard deviation sition of the inspected material̶C. spicatus was found chromosome number of the species was found x=9. The to have the diploid complement of 18 chromosomes with reports regarding to the number of diploid chromosomes a special feature containing an unpaired chromosome 2n=16 were recorded by Boehm in 1931 from C. spi- in 3 out of 5 analyzed plant materials with a percentage catus (syn. C. cylindricus) where all the chromosomes of 32 (Fig. 2a, b). The remarkably smallest chromo- are metacentric. Simmonds (1954) disclosed the diploid some can be recognized as B-chromosome. Our study chromosome number for the species as 2n=18. revealed that the investigated plants had 16 metacentric In C. spicatus, 32% of the studied cells were observed chromosomes, two submetacentric chromosomes show- with a B-chromosome in three out of five examined ing the karyotypic formula 2n=18=16 m+2sm, and an plant materials (Fig. 2b). The size of the B-chromosome unpaired metacentric B-chromosome (Table 1). No sat- was 0.85±0.27 µm with long arm 0.45±0.12 µm and ellite was spotted from the examined species.