Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(3): 37-45, 2024 Firenze University Press https://riviste.fupress.net/index.php/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-3007 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Jha, T.B., Halder, M. & Bhowmick, B.K. (2024). Giemsa-based chromosome staining and compara- tive fluorescent banding pattern in five valuable Indian plant species. Caryo- logia 77(3): 37-45. doi: 10.36253/caryo- logia-3007 Received: Sep 26, 2024 Accepted: Sep 29, 2024 Published: March 25, 2025 © 2024 Author(s). This is an open access, peer-reviewed article pub- lished by Firenze University Press (https://www.fupress.com) and distrib- uted, except where otherwise noted, under the terms of the CC BY 4.0 License for content and CC0 1.0 Uni- versal for metadata. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID TBJ: 0000-0003-0900-8167 MH: 0000-0002-8422-0814 BKB: 0000-0001-6029-1098 Giemsa-based chromosome staining and comparative fluorescent banding pattern in five valuable Indian plant species Timir Baran Jha1,*, Mihir Halder2, Biplab Kumar Bhowmick3 1 Department of Botany, Maulana Azad College, Rafi Ahmed Kidwi Road, Kolkata- 700013, West Bengal, India 2 Department of Botany, Barasat Government College, 10 KNC Road, Barasat, Kolkata- 700124, West Bengal, India 3 Department of Botany, Scottish Church College, 1 & 3, Urquhart Square, Manicktala, Azad Hind Bag, Kolkata-700006, West Bengal, India *Corresponding author. E-mail: tbjha2000@yahoo.co.in Abstract. This study presents repeatable enzymatic maceration and air drying (EMA)- based chromosome preparation methods in five valuable Indian plant species namely Allium cepa, Allium sativum, Nigella sativa, Trigonella foenum-graecum, and Aloe vera. Comparative fluorescent banding studies with two DNA base-specific fluorescent dyes have precisely unraveled the number, position, and patterns of secondary constric- tion of each species. Additionally, it has highlighted the fluorescent banding pattern of repetitive DNA sequences notably on two important constitutive heterochromatic sites like secondary and primary constrictions. The study has established that EMA-based fluorescent banding can provide valuable complementary information for modern genomics. The results are expected to enrich our knowledge of chromosome biology and crop genomics and inspire future academic and research endeavours. Keywords: CMA-DAPI banding, Giemsa staining, Allium cepa and Allium sativum, Aloe vera, Nigella sativa, Trigonella foenum-graecum. INTRODUCTION Plants have been serving humanity for centuries. They rely on their species-specific chromosomes not just to carry genetic information, but also for their precise maintenance, management, and transmission (Flavell 2021). Therefore, analyzing and conserving genetic diversity to safeguard their adaptive potential is crucial for valuable populations of wild and domesticat- ed species (Ainsworth 2022) in the era of climatic changes. Classical cytogenetics has provided foundational information on plant chromosomes for many decades (Guerra 2008; Razumova et al. 2023) in a cost-effective manner. On the other hand, molecular cytogenetics has opened the door to unraveling genetic diversity not only on precise chro- mosomal morphology but also on the chromosomal DNA composition of https://riviste.fupress.net/index.php/caryologia https://doi.org/10.36253/caryologia-3007 https://doi.org/10.36253/caryologia-3007 https://doi.org/10.36253/caryologia-3007 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode https://orcid.org/0000-0003-0900-8167 https://orcid.org/0000-0002-8422-0814 https://orcid.org/0000-0001-6029-1098 mailto:tbjha2000@yahoo.co.in 38 Timir Baran Jha, Mihir Halder, Biplab Kumar Bhowmick each population and species. Molecular cytogenetics has greatly benefited from the advancement of molecu- lar biology, revealing that chromosomes contain low- copy regulatory gene sequences, while large chunks of DNA are composed of diverse repetitive DNA sequenc- es throughout the length of the chromosomes (Liehr 2021). Some repetitive sequences are well conserved between species, while others define differences even between closely related species. In the 1970s, the intro- duction of the enzymatic maceration and air-drying (EMA) protocol for plant chromosome preparation (Kurata and Omura 1978), followed by Giemsa stain- ing, and the subsequent application of DNA base-spe- cific fluorochromes in plant chromosome research have helped in individual chromosome identification. These advancements have allowed a profound revision of the structure and function of repetitive DNA patterns in nucleolar organizing regions (NORs), centromeric, sub- telomeric, and telomeric regions in many plant species (Fukui et al. 1996; Moscone et al. 1996; Hizume 2015; Jha and Halder 2016; Yamamoto et al. 2019; Jha et al. 2021; Jha and Halder 2023). Keeping in mind the above-noted considerations the present studies have standardized the EMA-based protocol for chromosome preparation in some valu- able Indian species and populations of Allium cepa L. (Amaryllidaceae), Allium sativum L. (Amaryllidaceae), Nigella sativa L. (Ranunculaceae), Trigonella foenum- graecum L. (Fabaceae), and Aloe vera (L.) Burm. f. (Asphodelaceae). All of these plant species are highly valued for both daily consumption and medicinal pur- poses. Detailed and comparative chromosomal analysis has been carried out using non-fluorescent Giemsa and two contrasting fluorochromes DAPI (4’-6-diamidino- 2-phenylindole) and CMA (chromomycin A3 -CMA) staining. The standardized and repeatable protocol effectively produced numerous cytoplasm-free meta- phases and subsequent staining with non-fluorescent and fluorescent dyes, precisely detailed their morphol- ogy, and number and positions of secondary constric- tions. Additionally, it has highlighted the fluorescent banding pattern of repetitive DNA sequences notably on two important constitutive heterochromatic sites, secondary and primary constrictions. The results of this study are expected to instil interest in students, researchers, and breeders in their future academic and research endeavours, as well as conserve and explore the unique genetic diversity found in other family members of the studied materials. MATERIAL AND METHODS Plant materials The bulbs of two commercially available Allium spe- cies, Allium cepa L. and Allium sativum L., were col- lected and grown in wet sand for 48 hours in the dark. The plants of Aloe vera (L.) Burm. f were obtained from a local nursery in Kolkata, West Bengal and grown in the medicinal garden of the Maulana Azad College. The healthy roots of A. vera were directly harvested from potted plants. Seeds of Trigonella foenum-graecum L. were obtained from the local market, while seeds of Nigella sativa L. were collected from a farmer’s field in North 24 Parganas, West Bengal, and stored at 4°C. At least 20 seeds of each species were soaked overnight in water, then placed on moist filter paper and kept in the dark at temperatures between 16-22°C for germination. Experimental materials were used several times to har- vest roots for chromosome analysis. Chromosome preparation by enzymatic maceration and air-drying protocol To standardize the chromosome preparation using the EMA protocol, a minimum of ten healthy root tips meas- uring 0.7-1 cm in length were collected from germinating seeds, bulbs, or potted plants. Root tips of A. cepa, A. sati- vum and A. vera were treated with a saturated solution of Para Dichlorobenzene (PDB), Trigonella foenum-graecum with PDB plus Aesculin and Nigella sativa with 0.5% Col- chicine for 4-5 hours. Fixation was carried out with a 1:3 solution of acetic acid and methanol overnight and then stored at -20°C. The chromosome preparation was carried out following the EMA protocol established by Jha and Hal- der (2023), with some species-specific minor modifications. Chromosome staining with Giemsa, DAPI and CMA The air-dried slides of each species were stained with a phosphate buffer solution containing 2% Giemsa solu- tion (Merck, Germany) for 10-15 minutes to achieve con- sistent, effective, and optimal staining. After staining, the slides were rinsed three to four times with distilled water air dried with a blower, and finally used for chromosome analysis under a Carl Zeiss compound microscope. The best Giemsa-stained metaphase slides were destained in 70% methanol for 45 minutes, air-dried and subsequently re-stained with DAPI (0.1-0.2 µg mL-1 for 10-15 minutes) and CMA (0.1-0.8 mg mL-1 for 70-120 minutes), following Jha et al. (2021) with species-specific modifications. 39Giemsa-based chromosome staining and comparative fluorescent banding pattern in five valuable Indian plant species Chromosome analysis, documentation, and ideogram prep- aration A Carl Zeiss AxioLab A1 fluorescence microscope equipped with a CCD camera and computer software was used to examine, documentation, and karyotype analysis of cytoplasm-free metaphase chromosome preparations stained with Giemsa, DAPI, and CMA. Giemsa-stained slides were observed under bright field illumination, while DAPI- and CMA-stained slides were observed under a specific UV filter cassette using the Carl Zeiss Prog Res 2.3.3 software on a computer attached to the microscope. Images of metaphase chro- mosomes stained with Giemsa, DAPI, or CMA were captured with the help of a CCD camera for analysis. More than 25 cytoplasm-free mitotic metaphases with well-spread chromosomes displaying distinct karyomor- phometric features were chosen for each species. Docu- mentation and measurement of karyomorphometric fea- tures such as the lengths of the long and short arms, the positions of the centromeres, the average chromosome length (ACL), the total chromatin length (TCL), and DAPI and CMA signals were performed using Axiovision L.E.4 software. At least five chromosome morphometric data for each species were considered for the calculation of ACL and TCL, and the data was expressed as the mean ± stand- ard deviation (SD). Chromosome nomenclature was based on arm ratios (r = length of long arm/length of short arm) following Levan et al. (1964), and ideograms were pre- pared by evaluating morphometric data and organizing chromosome pairs in decreasing order of length. RESULTS The karyomorphological features of each plant spe- cies were determined from cytoplasm free well-spread mitotic metaphases and documented (Figures 1-4, and Table 1-2). Fluorescent staining with DAPI and CMA revealed distinct variability in banding patterns on the chromosomes (Figures 1, 3, Table 2). For the conveni- ence of interpretation, based on the nature of the fluo- rescent banding pattern, chromosomes were categorized into eight types, namely, type A with CMA+ve (positive) signal in the centromeric region, type B with DAPI+ve signal in the centromeric region, type C with CMA+ve signal at two terminal regions of chromosome, type D with CMA+ve signal at the secondary constriction region, type E with CMA+ve signal at the satellite region and two terminal regions of chromosome, type F CMA0 (neutral) / DAPI0 (neutral), type G with CMA+ve signals at the centromeric region as well as at the secondary constric- tion region and type H with DAPI+ve signals at centro- meric region and CMA+ve signals in the secondary con- striction region (Table 2). It was further confirmed that all CMA+ve bands were DAPI-ve and the DAPI+ve bands were CMA-ve. The unique karyotypes and fluorochrome banding patterns observed in each species are described as follows: Allium cepa L. (Amaryllidaceae) The karyotype in Allium cepa (2n=16), the age-old model plant material for chromosome studies, revealed the occurrence of five metacentric and three submeta- centric pairs of long chromosomes (Fig. 1a-c, Fig. 2a, Figure 1. Somatic metaphase chromosomes of Allium cepa (2n=16) stained correspondingly with Giemsa (a), DAPI (b) and CMA (c); Somatic metaphase chromosomes of Allium sativum (2n=16) stained correspondingly with Giemsa (d, g, j), DAPI (e, h, k) and CMA (f, i, l). The positions of two (d-f), three (g-i) and four (j-l) second- ary constrictions are indicated with black arrows in Giemsa-stained metaphases and corresponding DAPI negative signals are indicated with white arrows with round end in DAPI-stained metaphases. The positions of terminal CMA positive signals are indicated with white arrows in CMA-stained metaphases. Scale bars=10 µm. 40 Timir Baran Jha, Mihir Halder, Biplab Kumar Bhowmick Table 1). Out of three submetacentric pairs of chromo- somes, the 6th pair of chromosomes bears the second- ary constriction at terminal regions. Screening of over 25 metaphase plates stained with CMA fluorochrome confirmed bright CMA+ve signals at the terminal regions of all chromosomes (Type C, Fig. 1c). Notably, CMA+ve signal was also observed on the satellite of the 6th pair of chromosomes (Type E, Fig. 1c, 2a). Of the one pair of chromosomes with secondary constriction, one of the chromosomes showed a more intense CMA+ve signal on the satellite than the other homologue (Fig. 1c), corre- sponding with the difference in the size of this satellite in Giemsa-stained chromosomes (Fig. 1a). On the other hand, no AT-specific DAPI+ve signal was observed in any of the chromosomes of this germplasm of A. cepa (Fig. 1b). The banding pattern of A. cepa thus remains 14C+ 2E with CMA+ve and DAPI –ve signals (Table 2). Allium sativum L. (Amaryllidaceae) The studied germplasm of Allium sativum L. (Indi- an garlic) has 2n =16 chromosomes (Fig. 1d-l, Table 1), revealing three variant karyotypes with variable num- bers (2-4) of chromosomes with secondary constrictions (Fig. 2b-d, Table 2). However, the position of secondary constriction was always interstitial (Fig. 1d-l). The modal karyotype of this species is 10m+4sm+2sm.st (Fig. 2b), with the 6th pair of submetacentric chromosomes exhib- iting a secondary constriction region (Fig. 1d). In addi- tion, we have noted three chromosomes with secondary constrictions, i.e. on the 6th pair of both homologous chromosomes plus one chromosome of the 8th pair (Fig. 2c) in some metaphases. Moreover, very few metaphase plates also showed four chromosomes with secondary Figure 2. Somatic ideogram of Allium cepa (2n=16) with two sec- ondary constriction-bearing chromosomes (a); Somatic ideograms of Allium sativum (2n=16) with two (b), three (c) and four (d) chromosomes with secondary constrictions. The positions of CMA- positive signals are indicated with black bands. Scale bars=5 µm. Table 1. Karyomorphometric data in the five valuable Indian plant species. Species (Diploid chromosome number) CSR in μm (mean±SD) ACL in µm (Mean±SD) TCL in μm (mean±SD) Number of SAT chromosomes (pair number length-wise) Position of satellite Diploid Karyotype Allium cepa (2n=16) 10.9±1.20-19.34±2.66 16.93±2.64 241.09±9.85 2 (6th) T 10m+4sm+2sm.t Allium sativum (2n=16) 8.46±0.71-19.67±2.93 13.54±1.81 224.18±5.85 2 (6th) I 10m+4sm+2sm.st 3 (6th, 8th) I 10m+3sm+3sm.st 4 (6th, 8th, 1st) I 10m+2sm+3sm.st+1m.sm Nigella sativa (2n=12) 7.29±0.65-16.64±2.09 13.54±1.81 159.0.9±18.06 6 (2nd, 3rd, 6th) I 6m+4m.t+2t.sat Trigonella foenum-graecum (2n=16) 8.85±0.76-12.21±1.25 10.34±1.17 165.52±18.42 4 (2nd, 4th) I 2m+4sm+6st+2m.sm+2m.st Aloe vera (2n=14) 8.52±0.60-30.75±1.08 21.13±10.12 295.86±10.72 4 (1st, 3rd) T 4st+6sm+4sm.t CSR: Chromosome size range (range of absolute length of shortest to longest chromosome), ACL: Average length of chromosomes, TCL: Total diploid chromosome length, SAT: Satellite bearing chromosomes, T: Terminal, and I: Interstitial. http://sm.st 41Giemsa-based chromosome staining and comparative fluorescent banding pattern in five valuable Indian plant species constrictions, i.e. on the 6th pair of submedian homolo- gous chromosomes, on one of the 8th pair of submedian chromosomes, and one on the 1st pair of metacentric chromosomes (Fig. 2d; Table 1). Detection of satellite in two heteromorphic and non-homologous chromosomes, i.e., one submedian and one metacentric chromosome, was another interesting finding in this germplasm (Fig. 2d). Interestingly, different karyotype variants were not detected in different metaphases of the same root tip; they were detected in different root tips. In A. sativum, none of the 16 chromosomes showed any CMA+ve or DAPI+ve signals even after repeated trials with varying concentrations of fluorochromes and vary- ing incubation periods. The banding pattern of A. sati- vum thus remains 16F i.e. CMA0 / DAPI0 (Fig. 1e, f, h, i, k, l, 2b-d, Table 2). Nigella sativa L. (Ranunculaceae) The karyotype of N. sativa (2n=12) was character- ized by one pair of distinctly short telocentric chromo- somes and five pairs of long metacentric chromosomes (Fig. 3a-c, Table 1). The secondary constrictions were located on terminal regions of one pair of short chromo- somes (6th) and two pairs (2nd and 3rd) of long chromo- somes (Fig. 3a, Table 1). The modal karyotype of N. sati- va was determined as 6m+4m.t+2t.sat (Fig. 4a, Table 1). CMA-DAPI staining was performed on the same Giemsa-stained metaphase plates to confirm the exist- ence of secondary constrictions in three chromosome pairs (Fig. 3b-c). The staining revealed intense CMA+ve signals at secondary constriction regions that corre- sponded with DAPI-ve signals (Fig. 3b-c). No addition- al distinct DAPI/CMA positive/negative signals were found on any chromosomes (Fig. 3b-c) and the fluoro- chrome banding pattern was determined as 6D+6F (Fig. 4a, Table 2). Trigonella foenum-graecum L. (Fabaceae) In T. foenum-graecum (2n=16), we have identi- fied the presence of interstitial secondary constric- tions at the submetacentric and subtelocentric positions of the 2nd and 4th pairs of chromosomes, respectively (Fig. 3d-f, 4b). The modal karyotype was determined as 6st+4sm+2m+2m.sm+2m.st (Fig. 4b, Table 1). Fluores- cent staining revealed very distinct and intense CMA+ve and DAPI-ve signals in all chromosomes at the centro- meric regions (Fig. 3e-f). Additionally, the interstitial secondary constriction region of the 2nd and 4th pair of chromosomes also showed CMA+ve and DAPI-ve signals (Fig. 3e, f, Table 2). The fluorescent banding pattern in T. foenum-graecum was 12A+4G (Fig. 4b, Table 2). Aloe vera (L.) Burm. f. (Asphodelaceae) The karyotype in A. vera (2n=14) consists of three pairs of small (8.52-11.30 μm) submetacentric chromo- somes and four pairs of distinctly long (28.26-30.75 μm) chromosomes (Fig. 3g-i, Table 1). The primary constric- tions of the long chromosomes were located at the sub- metacentric or subtelocentric positions, with secondary constrictions at the terminal region of the 1st and 3rd pairs of long chromosomes. (Fig. 3g-i, Table 1). Fluorescence staining with different concentrations of DAPI and CMA enabled confirmation of chromo- somes with secondary constrictions and additional het- erochromatic bands in the karyotype. Very faint CMA+ve signals could be seen in two to three chromosomes at their secondary constriction regions (Fig. 3i, Table 2), Figure 3. Somatic metaphase chromosomes of Nigella sativa (2n=12) stained correspondingly with Giemsa (a), DAPI (b) and CMA (c); Somatic metaphase chromosomes of Trigonella foenum- graecum (2n=16) stained correspondingly with Giemsa (d), DAPI (e) and CMA (f); Somatic metaphase chromosomes of Aloe vera (2n=14) stained correspondingly with Giemsa (g), DAPI (h) and CMA (i). The positions of secondary constrictions are indicated with black arrows in Giemsa-stained metaphases. DAPI-negative and centromeric DAPI-positive signals are indicated with white arrows with round end and white arrowheads, respectively in DAPI-stained metaphases. CMA-positive signals are indicated with white arrows in CMA-stained metaphases. Scale bars=10 µm. http://m.sm http://m.st 42 Timir Baran Jha, Mihir Halder, Biplab Kumar Bhowmick corresponding to DAPI-ve signals (Fig. 3h). On the other hand, distinct and intense DAPI+ve signals were scored in all six small chromosomes i.e. in the 5th, 6th and 7th pairs (Fig. 3h, Table 2) and four out of eight long chro- mosomes. Of these four long chromosomes, one pair had secondary constrictions, and the other was subtelo- centric (Fig. 3h, 4c, Table 2). The distinctive CMA-DAPI banding pattern in A. vera was 8B+2H+3F+1D (Fig 4c, Table 2). DISCUSSION The current research has focused on standardizing chromosome processing using EMA-based preparation and a staining protocol that utilizes both fluorescent and non-fluorescent stains. The fluorescent banding in the species under study has revealed many new features, particularly in two important constitutive heterochro- matic sites: secondary and primary constrictions. Both of these sites are considered to be evolutionarily con- served with diverse functions. There are very few com- parative fluorescent banding studies available on seem- ingly common but highly valued plants. In a standard karyotype of any plant species, the nucleolus organizer region (NOR) acts as a chromosom- al marker for researchers and breeders. Guerra (2000) noted that secondary constriction regions are gener- ally GC-rich and display CMA+ve signals. The number and position of secondary constriction regions in the studied materials following the preparation of chromo- somes through classical cytogenetics is still a topic of debate (Sato 1980, 1981; Agarwal 1983; Das et al. 2001; Ghosh and Dutta 2006; Das et al. 2011; Martin et al. 2011; Najafi et al. 2013; Shaker et al. 2017). The present investigation confirms the number and positions of sec- ondary constrictions in all the studied species (Table Figure 4. Somatic ideograms of (a) Nigella sativa (2n=12) with six secondary constriction-bearing chromosomes, (b) Trigonella foenum-graecum (2n=16) with four secondary constriction-bear- ing chromosomes, and (c) Aloe vera (2n=14) with four secondary constriction-bearing chromosomes. The positions of CMA-positive and DAPI-positive signals are indicated with black and grey bands, respectively. Scale bars=5 µm. Table 2. Fluorescent banding patterns in the five valuable Indian plant species. Species (diploid chromosome number) Fluorescent banding patterns Total number of fluorescent signals (CMA+ve + DAPI-ve) Banding patternChromosome number (p: pair/ s: single) Position of the signals Type of signals Number of signals Allium cepa (2n=16) 1-8th (p) Terminal region CMA+ 32 34 14C+2E 6th (p) Satellite region CMA+ 2 Allium sativum (2n=16) 1st (s), 6th (p), 8th (s) Secondary constriction region CMA0 DAPI0 Nil Nil 16F Nigella sativa (2n=12) 2nd, 3rd, 6th (p) Secondary constriction region CMA+ 6 6 6D+6F Trigonella foenum- graecum (2n=16) 2nd, 4th (p) Secondary constriction region CMA+ 4 20 12A+4G 1-8th (p) Centromeric region CMA+ 16 Aloe vera (2n=14) 1st (p), 4-7th (p) Centromeric region DAPI+ 10 12-13 8B+2H+3F+1D 1st (p), 3rd (s) Secondary constriction region faint CMA+ 2-3 Fluorescent banding types- A: CMA-positive centromeric region, B: DAPI-positive centromeric region, C: CMA-positive terminal regions, D: CMA-positive secondary constriction region, E: CMA-positive terminal and secondary constriction regions, F: CMA- neutral and DAPI- neutral, G: CMA-positive centromeric and secondary constriction regions, H: DAPI-positive centromeric region and CMA-positive second- ary constriction region. 43Giemsa-based chromosome staining and comparative fluorescent banding pattern in five valuable Indian plant species 1). In Allium cepa and Allium sativum, distinct differ- ences in the number of chromosomes bearing second- ary constrictions and fluorescent banding patterns were noted. In A. cepa, we scored 34 CMA+ve signals includ- ing a pair of chromosomes with a CMA+ve satellite region (Fig. 1b-c, Table 2). In this Indian germplasm, we report heteromorphy concerning CMA+ve/DAPI-ve (GC-rich) band intensity or size in the 6th pair of chromosomes with satellite. Our results conform to Cortes et al. (1983) and Mancia et al. (2015) who have reported terminal bands in all the chromosomes of A. cepa through their C-banding and dual colour FISH studies, respectively. The genus Allium has a history of possible chromo- some rearrangements related to the distal and interstitial location of NORs in subgenera Cepa and Allium, respec- tively (Bhowmick et al. 2023). Numerical variations of secondary constrictions (2-6) have been reported in A. sativum (Sato et al. 1980; Cortes et al. 1983; Wajahatul- lah and Vahidy 1990). But for the first time, we report two to four interstitial secondary constrictions (Fig. 1d-l, Fig. 2b-d) in the Indian germplasm. In A. sativum, no CMA and DAPI-positive bands were observed in any chromosomes. It has been reported that chromo- somal CMA bands are generally limited for A. sativum (Maragheh et al. 2019; Bacelar et al. 2021). However, C-bands were reported in some chromosome pairs of A. sativum (Cortes et al. 1983; Yuzbasioglu 2004). The atyp- ical DNA banding pattern in general especially of the secondary constriction regions in A. sativum warrants further molecular analyses to unravel the structural complexity and uniqueness. The use of EMA-based chromosome processing and differential staining has confirmed the presence of three pairs of secondary constrictions for the first time in Nigella sativa. Our findings are consistent with the 45S rDNA-loci analysis conducted by Orooji et al. (2022) in a different germplasm of N. sativa. However, the observed variation in CMA staining intensity in the NOR regions needs to be addressed in the future at the DNA sequence level. While, in Trigonella foenum-graecum, fluorescent banding studies confirmed the presence of two pairs of interstitial secondary constrictions on the 2nd and 4th chromosome pairs. Similar results have been observed in other germplasms through fluorescent banding and rDNA hybridization studies (Ahmad et al. 1999; Santra et al. 2023). Fluorescent banding studies in Aloe vera reveal secondary constrictions at the terminal region of 1st and 3rd pairs of long chromosomes (Table 2). How- ever, CMA+ve/ DAPI-ve signals were obtained distinctly in one pair of secondary constricted chromosomes and the other pair remains indistinct and difficult to determine in many metaphases. Adams et al. (2000) reported vari- ability in rDNA sites within the genus Aloe and reported three pairs of rDNA sites in A. vera. In comparative studies of centromeric sites, three types of DNA sequence organization have been observed in the studied species: CMA+ve/DAPI-ve (GC rich), DAPI+ve/CMA-ve (AT-rich), and CMA0/ DAPI0 (GC-AT neutral). CMA+ve centromeric sites have not been reported in many plant species. Mondin et al. (2011) reported it in Crotalaria juncea (2n=16). Than et al. (2017) reported CMA+ve centromeric sites in all the chromosomes of Swertia nervosa (2n=26), only in one pair in S. chirayita (2n=26), but not a single centromeric region of S. bimaculata (2n=26) chro- mosomes. In Trigonella, intense CMA+ve signals have been found in the centromeric regions of all chromo- somes in the present study. A similar report has been published by Santra et al. (2023) in Trigonella. Further f luorescent banding studies in different plant species are required to unravel DNA sequence patterns. Fur- ther f luorescent banding studies in Trigonella spe- cies and populations are equally necessary to confirm whether the CMA+ve GC-rich centromeric nature is global or local in Trigonella. On the other hand, a well-established bimodal karyotype in Aloe vera with eight long and six small chromosomes (2n=14) showing unique DAPI+ve/CMA- ve fluorescent banding patterns in all six small and four long chromosomes is reported for the first time in this species. It is recognized that the centromere in every eukaryotic chromosome is a multifunctional dynamic complex. Despite mediating the evolutionarily con- served function of directing chromosome segregation, they show surprising diversity in their DNA sequence organisation (Tong et al. 2019; Barbosa et al. 2022). DNA sequence analysis in rice, maize, and Arabidop- sis has shown that centromeres are composed of highly repetitive DNA sequences with considerable size varia- tion and sequence divergence (Ma et al. 2007) but rare- ly has it been characterized as GC- or AT-rich or GC– AT neutral regions. On the other hand, the fluorescent banding patterns directly observed on the metaphases in highly conserved centromeric sites of Trigonella, Aloe, Nigella and Allium revealed their contrasting spe- cies and even chromosome-specific DNA divergence and necessitate further fluorescent banding and DNA sequencing studies in the future. We encourage further analysis of fluorescent banding in other plant species to gain a better understanding of the nature of DNA organization in secondary and centromeric regions. We hope that our findings will inspire researchers and scientists to come up with new ideas for studying chro- mosomes in plants. 44 Timir Baran Jha, Mihir Halder, Biplab Kumar Bhowmick CONCLUSION The use of a repeatable EMA-based protocol for comparative fluorescent banding can be applied to any plant species with minor modifications. Recent stud- ies on five common but valuable plant species have pro- vided interesting and thought-provoking information on the nature of repetitive DNA sequences in two consti- tutive heterochromatin sites directly on the metaphase plates. The research has shown that EMA-based fluores- cent banding can offer valuable complementary infor- mation for modern genomics. The results are expected to enhance our understanding of chromosome biology and crop genomics and to inspire future academic and research endeavours. ACKNOWLEDGEMENTS In honour of his mentor and teacher, the late Prof. A. K. Sharma, TBJ dedicates this paper to his birth cen- tenary year. He would like to acknowledge Dr S. Dutta, the Principal, and Dr D. Mukhopadhya, the Head of the Department of Botany at Maulana Azad College, for providing basic facilities. MH expresses gratitude to the Principal of Barasat Government College, Kolkata, for his active support and encouragement in research activi- ties. BKB is thankful to the Principal of Scottish Church College for encouraging research activities. REFERENCES Adams SP, Leitch IJ, Bennett MD, Chase MW, Leitch AR. 2000. Ribosomal DNA evolution and phylogeny in Aloe (Asphodelaceae). Ame J Bot. 87(11): 1578–1583. Agarwal K. 1983. Cytological studies in the genus Trigo- nella Linn. Cytologia 48(4):771–779. https://doi. org/10.1508/cytologia.48.771 Ahmad F, Acharya SN, Mir Z, Mir PS. 1999. Localiza- tion and activity of rRNA genes on fenugreek (Trigo- nella foenum-graecum L.) chromosomes by fluores- cent in situ hybridization and silver staining. Theor Appl Genet. 98:179–185. https://doi.org/10.1007/ s001220051056 Ainsworth D. 2022. UN convention on biological diver- sity (COP 15) held in Montreal Canada. Bacelar PAA, Feitoza LL, Valente SES, Gomes RLF, Mar- tins LV, Almeida PM, Silva VB, Lopes ACA, Car- valh R, Peron AP. 2021. Variations in heterochro- matin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L.). Braz J Biol. 83: e243514. https://doi. org/10.1590/1519-6984.243514 Barbosa AC, Xu Z, Karari K, Williams W, Hauf S, Brown WR. 2022. Mutation and selection explain why many eukaryotic centromeric DNA sequences are often A+ T rich. Nu Acid Res. 50(1):579–596. https://doi. org/10.1093/nar/gkab1219 Bhowmick BK, Sarkar S, Roychowdhury D, Patil SD, Lekhak MM, Ohri D, Rao SR, Yadav SR, Verma RC, Dhar MK, Raina SN. 2023. Allium cytogenetics: a critical review on the Indian taxa. Comp Cytogenet. 17:129. 10.3897/CompCytogen.17.98903 Cortes F, Gonzalez-Gil G, Hazen MJ. 1983. C-banding and sister chromatid exchanges in three species of the genus Allium (A. cepa, A. ascalonicum and A. sativum). Caryologia 36(3):203–210. https://doi.org/1 0.1080/00087114.1983.10797661 Das A, Mukherjee P, Ghoroi A, Jha TB. 2011. Compara- tive karyomorphologycal analyses of in vivo and in vitro grown plants of Aloe vera l. Burm. F. Nucleus 53(3):89–94. Das AB, Mohanty S, Das P. 2001. Cytophotometric esti- mation of 4C DNA content and karyotype analysis in ten cultivars of Trigonella foenum-graecum. Iran J Bot. 9(1):1–9 Flavell RB. 2021. Perspective: 50 years of plant chromo- some biology. Plant Physiol. 185(3):731-53. https:// doi.org/10.1093/plphys/kiaa108 Fukui K. 1996. Plant chromosomes at mitosis. In: Fukui K, Nakayama S, editors. Plant chromosomes: labora- tory methods. Boca Raton (BR): CRC Press; p. 1–17. Ghosh A, Datta AK. 2006. Karyotyping of Nigella sativa L. (black cumin) and Nigella damascena L. (love-in-a- mist) by image analyzing system. Cytologia 71(1):1–4. Guerra M, Santos KGBD, Silva AEB, Ehrendorfer F. 2000. Heterochromatin banding patterns in Rutace- ae-Aurantioideae– a case of parallel chromosomal evolution. Am J Bot. 87(5):735–747. https://doi. org/10.1159/000121083 Guerra M. 2008. Chromosome numbers in plant cytotax- onomy: concepts and implications. Cytogenet Genom Res. 120(3–4):339–350. Hizume M. 2015. Fluorescent banding pattern in chromo- somes of Tsuga forrestii and T. Sieboldii Pinaceae. Chro- mo Bot. 10(3):95–100. https://doi.org/10.3199/iscb.10.95 Jha TB, Bhowmick BK, Roy P. 2021. Analysis of CMA- DAPI bands and preparation of fluorescent karyo- types in thirty Indian cultivars of Lens culinaris. Car- yologia 74(2):65–77. Jha TB, Halder M. 2016. Searching chromosomal land- marks in Indian Lentils through EMA-based Giemsa staining method. Protoplasma 253:1223–1231. https://doi.org/10.1508/cytologia.48.771 https://doi.org/10.1508/cytologia.48.771 https://doi.org/10.1007/s001220051056 https://doi.org/10.1007/s001220051056 https://doi.org/10.1590/1519-6984.243514 https://doi.org/10.1590/1519-6984.243514 https://doi.org/10.1093/nar/gkab1219 https://doi.org/10.1093/nar/gkab1219 https://doi.org/10.1080/00087114.1983.10797661 https://doi.org/10.1080/00087114.1983.10797661 https://doi.org/10.1093/plphys/kiaa108 https://doi.org/10.1093/plphys/kiaa108 https://doi.org/10.1159/000121083 https://doi.org/10.1159/000121083 https://doi.org/10.3199/iscb.10.95 45Giemsa-based chromosome staining and comparative fluorescent banding pattern in five valuable Indian plant species Jha TB, Halder M. 2023. Evaluation of karyotype diver- sity in Indian traditional aromatic rice cultivars through EMA-based non-fluorescent Giemsa and fluorescent DAPI staining. Genet Resour Crop Evol. 1–22. https://doi.org/10.1007/s10722-023-01696-4 Kurata N, Omura T. 1978. Karyotype analysis in rice I. A new method for identifying all chromosome pairs. Jpn J Genet. 54:251–255. https://doi.org/10.1266/jjg.53.251 Levan A, Fredga K, Sandberg AA. 1964. Nomenclature for centromeric position on chromosomes. Heredi- tus. 52:210–220. Liehr T. 2021. Molecular cytogenetics in the era of chro- mosomics and cytogenomic approaches. Front Genet. 12:720507. Ma J, Wing RA, Bennetzen JL, Jackson SA. 2007. Plant centromere organization: a dynamic structure with conserved functions. Trends Genet 23(3):134-139. Mancia FH, Sohn SH, Ahn YK, Kim DS, Kim JS, Kwon YS, Kim CW, Lee TH, Hwang YJ. 2015. Distribution of various types of repetitive DNAs in Allium cepa L. based on dual-colour FISH. Hortic Environ Biotech- nol. 56(6):793–799. https://doi.org/10.1007/s13580- 015-1100-3 Maragheh FP, Janus D, Senderowicz M, Haliloglu K, Kolano B. 2019. Karyotype analysis of eight cultivat- ed Allium species. J Appl Genet. 60(1):1–11. https:// doi.org/10.1007/s13353-018-0474-1 Martin E, Akan H. Ekici M, Aytac Z. 2011. Karyotype analyses of ten sections of Trigonella (Fabaceae). Comp Cytogenet. 5(2):105. 10.3897/compcytogen. v5i2.969 Mondin M, Aguiar-Perecin ML. 2011. Heterochromatin patterns and ribosomal DNA loci distribution in dip- loid and polyploid Crotalaria species (Leguminosae, Papilionoideae), and inferences on karyotype evolu- tion. Genome 54:718–726. https://doi.org/10.1139/ g11-034 Moscone EA, Lambrou M, Ehrendorfer F. 1996. Fluores- cent chromosome banding in the cultivated species of Capsicum (Solanaceae). Plant Syst Evol. 202:37–63. https://doi.org/10.1007/BF00985817 Najafi S, Anakhatoon EZ, Birsin MA. 2013. Karyotype characterisation of reputed variety of fenugreek (Trig- onella foenum-graecum) in West Azerbaijan-Iran. J Appl Biol Sci. 7(1):23–26. Orooji F, Mirzaghaderi G, Kuo YT, Fuchs J. 2022. Vari- ation in the number and position of rDNA loci contributes to the diversification and speciation in Nigella (Ranunculaceae). Front Plant Sci. 13:917310. https://doi.org/10.3389/fpls.2022.917310 Razumova OV, Alexandrov OS, Bone KD, Karlov GI, Divashuk MG. 2023. Sex chromosomes and sex determination in dioecious agricultural plants. Agron 13(2):540. https://doi.org/10.3390/agronomy13020540 Santra I, Biswas D, Ghosh B. 2023. Chromosomal charac- terization mediated by karyomorphological analysis and differential banding pattern in fenugreek (Trigo- nella foenum-graecum L.): a neglected legume. Cary- ologia 76(3):63–70. https://doi.org/10.36253/caryolo- gia-2159 Sato S, Hizume M, Kawamura S. 1980. Relationship between secondary constrictions and nucleolus organizing regions in Allium sativum chromosomes. Protoplasma 105:77–85. https://doi.org/10.1007/ BF01279851 Sato S. 1981. Cytological studies on the satellited chro- mosomes of Allium cepa. Caryologia 34(4):431–440. https://doi.org/10.1080/00087114.1981.10796911 Shaker SS, Mohammadi A, Shahli MK. 2017. Cytological studies on some ecotypes of Nigella sativa L. in Iran. Cytologia 82(2):123–126. https://doi.org/10.1508/cyt- ologia.82.123 Than MMM, Samaddar T, Bhowmick BK, Jha S. 2017. Fluorescent chromosome banding and genome size estimation in three species of Swertia. Cytologia 82(5):513–520. Tong P, Pidoux AL, Toda NR, Ard R, Berger H, Shukla M, Torres-Garcia J, Müller CA, Nieduszynski CA, Allshire RC. 2019. Interspecies conservation of organisation and function between nonhomologous regional centromeres. Nat Commun. 10(1):2343. htt- ps://doi.org/10.1038/s41467-019-09824-4 Wajahatullah MK, Vahidy AA. 1990. Karyotyping and localization of nucleolar organizer regions in Garlic, Allium sativum L. Cytologia. 55(3):501–504. https:// doi.org/10.1508/cytologia.55.501 Yamamoto M, Takeuchi M, Nashima K, Yamamoto T. 2019. Enzyme maceration, fluorescent staining, and FISH of rDNA of Pineapple (Ananas comosus (L.) Merr.) chromosomes. J Hortic. 88(4):455–461. Yuzbasioglu D. 2004. Karyotyping, C- and NOR banding of Allium sativum L. (Liliaceae) cultivated in Turkey. Pak J Bot. 36(2):343–349. https://doi.org/10.1007/s10722-023-01696-4 https://doi.org/10.1266/jjg.53.251 https://doi.org/10.1007/s13580-015-1100-3 https://doi.org/10.1007/s13580-015-1100-3 https://doi.org/10.1007/s13353-018-0474-1 https://doi.org/10.1007/s13353-018-0474-1 https://doi.org/10.1139/g11-034 https://doi.org/10.1139/g11-034 https://doi.org/10.1007/BF00985817 https://doi.org/10.3389/fpls.2022.917310 https://doi.org/10.3390/agronomy13020540 https://doi.org/10.36253/caryologia-2159 https://doi.org/10.36253/caryologia-2159 https://doi.org/10.1007/BF01279851 https://doi.org/10.1007/BF01279851 https://doi.org/10.1080/00087114.1981.10796911 https://doi.org/10.1508/cytologia.82.123 https://doi.org/10.1508/cytologia.82.123 https://doi.org/10.1038/s41467-019-09824-4 https://doi.org/10.1038/s41467-019-09824-4 https://doi.org/10.1508/cytologia.55.501 https://doi.org/10.1508/cytologia.55.501 The genome of the southern short-horned tree dragon Acanthosaura meridiona Trivalairat, Sumontha, Kunya & Chaingkul, 2022 (Squamata, Draconinae) was analyzed using classical and molecular techniques to identify and study its chromosomal and repetitive ele Praween Supanuama, Sittisak Jantaratb*, Thaintip Kraipromb, Somsak Buathipb, Sarun Jumrusthanasanc, Sarawut Kaewsric, Nattasuda Donbunditd, Phichaya Buasriyote, Weera Thongnetrf, Sumalee Phimphang, and Alongklod Tanomtongd Amelioration strategy of saline stress in wheat with salicylic acid: a review Syeda Afia Fairoj1,†, Uttam Kumar Ghosh1,†, Md. 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