Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(3): 63-70, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2159 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Santra, I., Biswas. D., & Ghosh, B. (2023). Chromosomal char- acterization mediated by karyomor- phological analysis and differential banding pattern in fenugreek (Trigo- nella foenum-graecum L.): a neglected legume. Caryologia 76(3): 63-70. doi: 10.36253/caryologia-2159 Received: June 2, 2023 Accepted: October 31, 2023 Published: February 29, 2024 Copyright: © 2023 Santra, I., Biswas. D., & Ghosh, B. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distrib- uted under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, pro- vided the original author and source are credited. 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 IS: 0000-0001-7882-0090 DB: 0000-0002-3449-8543 BG: 0000-0002-4396-2088 Chromosomal characterization mediated by karyomorphological analysis and differential banding pattern in fenugreek (Trigonella foenum-graecum L.): a neglected legume Indranil Santra, Diptesh Biswas, Biswajit Ghosh* Plant Cytogenetics Laboratory, Post Graduate Department of Botany, Ramakrishna Mis- sion Vivekananda Centenary College, Rahara, Kolkata -700118, India *Corresponding author. E-mail: ghosh_b2000@yahoo.co.in Abstract. Fenugreek or Trigonella foenum-graecum L. is a commercially important yet neglected crop of the family Fabaceae, with potent medicinal applications, and can treat several diseases as well. Conventional breeding studies for higher yields of com- mercial crops largely depend on chromosomal information of the particular species. Despite a number of cytological research being conducted on T. foenum-graecum, a complete characterization of its chromosomes has not been achieved due to the limita- tions of traditional karyotype analysis methods. A range of chromosomal markers are advantageous to characterize at full extent and identify individual chromosomes rather than relying on only physical metrics. Thus, in this study, in addition to giemsa stain- ing, other approaches like fluorochrome and silver staining were used for the precise karyomorphological analysis of this species. Enzyme maceration and air drying (EMA) based fluorochrome banding with GC-specific stain Chromomycin A3 (CMA), and AT-specific stain 4’,6-diamidino-2-phenylindole (DAPI) applied for the first time for chromosome characterization. The results showed 2n = 16 chromosomes in metaphase cells, with karyotype formula of 2m+6sm. The unique banding pattern observed in the CMA/DAPI and AgNOR staining highlights the AT and GC-rich regions as well as the nucleolar organizer regions (NORs). All this crucial information can further assist in conducting breeding studies of more precision with simultaneously encouraging simi- lar studies that need to be done in other unexploited species of importance. Keywords: Trigonella foenum-graecum, Karyotype, CMA-DAPI, AgNOR, Fenugreek. INTRODUCTION Fenugreek (Trigonella foenum-graecum L.) belongs to the family Fabace- ae and has been consumed by the human race as food, spices and medicine since ancient times; nevertheless, it is still neglected from a global perspec- tive (Mikić 2015). The term “fenugreek” is derived from the Greek language, which translates to “Greek hay,” offering a glimpse into the plant’s histori- cal usage as a forage crop. The plant is cultivated in various regions, includ- ing India, Pakistan, Mediterranean Europe, Australia, and North America http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2159 https://doi.org/10.36253/caryologia-2159 http://www.fupress.com/caryologia http://www.fupress.com/caryologia https://orcid.org/0000-0001-7882-0090 https://orcid.org/0000-0002-3449-8543 https://orcid.org/0000-0002-4396-2088 mailto:ghosh_b2000@yahoo.co.in 64 Indranil Santra, Diptesh Biswas, Biswajit Ghosh (Acharya et al. 2008). India is a preeminent producer of fenugreek, claiming a staggering 80% of the global production (Rasheed et al. 2015). In addition to its culi- nary applications, the seeds and leaves of fenugreek have been utilized in traditional medicine to treat a plethora of conditions such as hyperglycemia, cardiovascular dis- ease, neurological disorders, pulmonary fibrosis, obesity, asthma, and inflammation. Fenugreek also possesses a large variety of nutri- tional compounds that are important for basic mainte- nance of biological systems. In general the fenugreek seeds contains 58% carbohydrates, 23-26% proteins, 0.9% fats and 25% fibers (Wani et al. 2018; Syed et al. 2020). Different kinds of minerals for example potas- sium (603 mg/100 g), magnesium (42 mg/100 g), calci- um (75 mg/100 g), zinc (2.4 mg/100 g), manganese (0.9 mg/100 g), copper (0.9 mg/100 g) and iron (25.8 mg/100 g) can be found in T. foenum-graecum. Vitamin C (220 mg/100 g) and β carotene (19 mg/100 g) are also present in higher amounts in fenugreek (Al-Jasass and Al-Jasser 2012; Wani et al. 2018). In addition, fenugreek contains several nutritionally valuable flavonoids such as querce- tin, luteolin, vitexin, 7, 4-dimethoxy flavanones, kaemp- ferol, tricin, and naringenin (Petropoulos 2002). Impor- tant amino acids including aspartic acid, glutamic acid, leucine, tyrosine, phenylalanine and free amino acid (2S, 3 R, 4S)-4-hydroxyisoleusine are abundantly present in fenugreek (Syed et al. 2020). In a study fenugreek seeds have been found to contain greater amounts of protein with better amino acid profile than soybean protein iso- late (Feyzi et al. 2002). Karyotyping is the process of classifying the chro- mosomal makeup of a cell by examining the number, size, and structure of each chromosome, which can pro- vide insights into the relationship between different spe- cies (Levin 2002). It is a commonly used technique in crop plant research for various purposes, such as charac- terizing cultivars, linking genetic and physical maps, and studying the evolutionary relationships among different species (de Moraes et al. 2007). Despite its utility, karyo- typing is often hindered by the scarcity of chromosome markers, which makes it challenging to identify individ- ual chromosomes (She and Jiang 2015). The utilization of traditional staining techniques can assist in examining the shape, size, and number of chromosomes, but it falls short of being able to differentiate between chromosomes that have similar physical characteristics (Shabir et al. 2017). In order to address the difficulty in distinguish- ing morphologically similar chromosomes, a number of chromosome banding techniques have been developed which offer a significant advantage for the identification of chromosomes and karyotyping (Andras et al. 2000). Chromosome staining with the combination of both chromomycin A3 (CMA) and 4’,6-diamidino-2-phe- nylindole (DAPI) fluorochromes has been widely used as a method to distinguish chromosome bands (Guerra 2000). CMA and DAPI, due to their proclivity for bind- ing to GC- and AT-rich sequences, respectively, allow for the discernment of various forms of heterochromatin as GC-abundant (DAPI-ve/CMA+ve), AT-abundant (DAPI+ve/ CMA-ve), or AT/GC-balanced (DAPI neutral/CMA neu- tral) bands (Barros e Silva and Guerra 2010). Nucleolar organizer regions (NORs) are another excellent chromo- some landmark effective in chromosomal characteriza- tion. The localization of NORs serves as a valuable mark- er for identifying chromosomes, offering a precise and dependable method of characterizing them (Maragheh et al. 2019). The presence and number of NORs in a cell can help distinguish between different types of chromosomes and provide important information for karyotyping. Cytological studies using karyotype analysis have been conducted for an extended period in different spe- cies and cultivars of fenugreek, having somatic chromo- some number 2n = 16 (Table 1). The karyotype reports concludes that any of the available species of the Foe- num-graecum section cannot be considered as the wild progenitor of fenugreek (Ladizinsky and Vosa 1986). The previous studies have been primarily limited to conven- tional karyotype analysis, with little emphasis placed on documenting and disseminating the findings (Table 1) (Agarwal and Gupta 1983; Bairiganjan and Patnaik 1989; Martin et al. 2011; Najafi et al. 2013). As far as our knowledge extends, the application of advanced differen- tial chromosome banding techniques such as CMA and DAPI has not been previously employed in the study of T. foenum-graecum. In light of this deficiency, the present study aims to fill this gap by utilizing these advanced techniques, in conjunction with silver staining (AgNOR), to perform a comprehensive characterization of the chro- mosomal structure of this species. The comprehensive characterization of chromosomes plays a crucial role in breeding programs. This process provides important information that enables breeders to make informed mat- ing decisions, leading to the production of offspring that possess both desirable traits and optimal health. MATERIAL AND METHODS Somatic chromosome preparation Seeds of T. foenum-graecum have been collected from the cultivated fields of Sainthia, Birbhum (24°00’55.9”N 87°44’09.4”E) West Bengal. The growing roots from ger- minated seeds of T. foenum-graecum were taken for 65Chromosomal characterization mediated by karyomorphological analysis and differential banding pattern in fenugreek chromosome preparation. Chromosomes were prepared following Santra et al. (2020) with minor modifica- tions. Roots were pretreated with 0.5 g L-1 8-hydroxyqui- noline solution at 16 °C for 6 h and then fixed in acetic acid:methanol solution (1:3) overnight. Digestion of the cell wall was performed with an enzyme mixture con- taining 1% cellulase (Onozuka-RS, Sigma, USA), 0.5% pectolyase (Sigma, USA), and 0.75% macerozyme (Serva, Germany) in a sodium citrate buffer (pH 4.6) at 37 °C for 90 mins. After washing with the same buffer twice, the root tip was broken down into small pieces on a clean slide with the addition of freshly prepared fixative. The slide was air-dried for at least 24 h before staining. Giemsa staining The chromosomes on the air-dried slide were first- ly stained with 2% giemsa solution in phosphate buffer, with a ratio of 1:15 (pH 6.8), followed by rinsing with distilled water and analyzed under a microscope. Photo- micrographs were taken with an AxioCam ICc 5 cam- era and ZEN application suite. Individual chromosomes were measured with AxioVision 4.9.1 and categorized based on the arm ratio following Levan et al. (1964). CMA and DAPI double staining Prior to simultaneous f luorochrome staining, with CMA and DAPI, the giemsa stained slides were destained with 70% methanol for 15 mins and air-dried. After preincubation of the slides in McIlvaine buffer (pH 7.0) for 10 mins, chromosomes were stained with 0.2 µg mL-1 DAPI solution for another 10 mins in the dark. After DAPI staining, slides were preincubated in McIl- vaine buffer (pH 7.0) supplemented with 5 mM MgCl2 and air dried. CMA staining was done with 0.25 mg mL-1 CMA solution for 60 min in the dark. After a short rinse in the same buffer, slides were mounted with 50% glycerol containing 5 mM MgCl2 and kept at 4 °C for 72 hrs before further analysis. Chromosomes were analyzed under the fluorescent microscope Zeiss Axio Scope A1 equipped with CMA and DAPI-specific filter cassettes. AxioCam ICc 5 and ZEN application suite were used to take the suitable photomicrographs. The karyogram has been carried out using Adobe Photoshop CS6. Silver staining In this study, the AgNOR staining was performed using the Ag-I procedure by Bloom and Goodpasture (1976), with a modification introduced by Kodama et al. (1980) of using nylon cloth instead of coverslips. Silver nitrate solution was added to slides, placed in moisture- proof plastic containers and covered with nylon mesh. To keep the environment moist, distilled deionized water is placed at the bottom of the containers, away from the slides. The slides are left to incubate in the water bath for 48 h at 45 °C. The NOR region appeared as dark brown color bands over light brown chromo- some arms. Table 1. Previous chromosome reports in Trigonella foenum-graecum. Sl. No. Chromosome counts Karyotype Symmetry/Asymmetry ReferenceGametophytic (n) cells Sporophytic (2n) cells 1. 8 16 1scAsm+5Asm+1Bm+1Csm Asymmetrical Agarwal and Gupta (1983) 2. 8 16 – – Laxmi et al. (1983) 3. – 16 – Asymmetrical Ladizinsky and Vosa (1986) 4. 8 16 – – Arya et al. (1988) 5. – 16 1m+5sm+2st Asymmetrical Bairiganjan and Patnaik (1989) 6. – 16 A2B12D2 – Kar and Sen (1991) 7. – 16 – – Jahan et al. (1994) 8. – 16 – – Ahmed et al. (1999) 9. – 16 – – Das et al. (2000) 10. – 16 – Symmetrical Das et al. (2001) 11. – 16 – Symmetrical Das et al. (2002) 12. – 16 2m+6sm – Martin et al. (2011) 13. – 16 10sm + 4smsat + 2m – Najafi et al. (2013) 14. – 16 – – Ranjbar and Zahra (2016) 66 Indranil Santra, Diptesh Biswas, Biswajit Ghosh RESULTS In this analysis with Trigonella foenum-graecum, more than 40 root tips were initially studied through giemsa staining, which confirmed that the somatic cells of the present cultivar contain 2n =16 chromosomes (Fig. 1a). Additionally, differential chromosome banding with CMA, DAPI and AgNOR have also been performed in metaphase as well as in prometaphase chromo- somes (Fig. 1b-f). The somatic chromosomes are small to medium in size and range between 4.70 to 5.92 μm. Individual chromosome sizes, arm ratio, and the cen- tromeric index has been mentioned in Table 2. Analysis through detailed karyomorphological studies revealed two pairs with median (m) to nearly median primary constriction and six pairs of chromosomes having sub- median (sm) primary constriction (Fig. 2a-d). Thus, the karyotype formula is 2m+6sm (Fig. 2d). Secondary con- strictions are also present in the long arm of one pair of metacentric chromosomes (pair 1) and in the short arm of one pair of submetacentric chromosomes (pair 4) (Fig. 2b,c). The secondary constrictions are interca- lary in position. The karyotype is symmetric and falls into 3A category of Stebbins’s (1971) classification. Later, fluorochrome staining with CMA and DAPI, revealed all eight pairs of chromosomes with bright, distinct and scorable CMA+ve bands, in their primary constriction (Fig. 2c). DAPI mostly stained the somatic metaphase chromosomes uniformly, however a single DAPI+ve band has been found in the chromosome pair 4 (Fig. 2b), in the intercalary position of short arm, colocalized with a CMA-ve band. DAPI-ve bands have been detected to be colocalized with the CMA+ve bands (Fig. 2b,c). Besides the single DAPI+ve band found in chromosome 4, several DAPI-brilliant regions were found in the prometaphase chromosomes (Fig. 1f), which also showed correspond- ing CMA-ve bands (Fig. 1e). However, in condensed meta- Figure 1. Differential chromosome banding in the somatic cells of Trigonella foenum-graecum. (a) giemsa stained metaphase plate; (b) silver staining (arrows indicate AgNOR bands); (c) CMA stained metaphase plate (arrows indicate CMA bands); (d) DAPI stained metaphase plate (arrow indicates DAPI bands); (e-f) CMA and DAPI stained prometaphase chromosomes. Scale bars of 5 µm. Table 2. Chromosome parameters and banding patterns in Trigonella foenum-graceum. Chromosome number S (µm) L (µm) Total (µm) Arm ratio Centromeric index Chromosome type* CMA bands (+/-) DAPI bands (+/-) AgNOR bands (+/-) 1 2.575 ± 0.019 3.350 ± 0.043 5.925 ± 0.053 1.301 0.434599 m + - + 2 1.789 ± 0.005 4.041 ± 0.005 5.830 ± 0.007 2.259 0.306872 sm + - - 3 1.527 ± 0.016 3.961 ± 0.010 5.487 ± 0.008 2.595 0.278186 sm + - - 4 1.886 ± 0.024 3.587 ± 0.007 5.473 ± 0.018 1.902 0.344541 sm +/- +/- + 5 1.388 ± 0.014 3.526 ± 0.012 4.914 ± 0.003 2.542 0.282366 sm + - - 6 1.231 ± 0.015 3.552 ± 0.018 4.783 ± 0.032 2.885 0.315542 sm + - - 7 1.5 ± 0.002 3.253 ± 0.004 4.753 ± 0.005 2.169 0.406730 sm + - - 8 1.912 ± 0.004 2.789 ± 0.002 4.701 ± 0.003 1.459 0.257422 m + - - *m = metacentric, sm = submetacentric. Total Chromatin Length (TCL) = 41.866 µm. 67Chromosomal characterization mediated by karyomorphological analysis and differential banding pattern in fenugreek phase chromosomes, these regions are found to be either dispersed or not clearly visible. Lastly, AgNOR staining specifically stained intercalary positions of chromosomes 1 and 4 (Fig. 1b). Thus, T. foenum-graecum chromo- somes can be identified and characterized based on the number and position of the CMA+ve/ DAPI-ve/ AgNOR bands (Fig. 2d). DISCUSSION According to Hutchinson (1964), the genus Trigo- nella is one of the six genera of the tribe Trifoliae and subtribe Trigonellinae. The genus Trigonella consists of approximately 134 species, which are found all over the world. These species can be diploid or polyploid, and there is evidence to suggest that their basic chromosome number could be x = 7, 8, or 9, as reported by different studies over the years. (Biddak 1996; Martin et al. 2011; Sharghi et al. 2020). The species T. foenum-graecum L. with basic chromosome number 8 (2n = 16) comes under the section Foenum-graecum along with eight other spe- cies (Basu 2023). Karyotype studies, chromosome band- ing and Fluorescent In Situ Hybridization techniques have depicted finer variation in species and cultivars of T. foenum-graecum L. (Agarwal and Gupta 1983; Ahmed et al. 1999; Das et al. 2000). T. foenum-graecum, in the present study shows 2n = 16 chromosomes in the somatic cell with the basic chromosome number x = 8 (Fig. 2d). The present study revealed the size of the somatic chro- mosomes was within a moderate range, ranging from 4.70-5.92 µm (Table 2). The karyotype formula, which is used to describe the number and appearance of chro- mosomes in a cell, was determined to be 2m+6sm. These findings were consistent with previous studies, indicat- ing a similarity in the chromosome size and formula between the present investigation and prior research (Martin et al. 2011). The process of enzymatic maceration of plant cells helps to prepare the chromosomes in a way that enables clear and unobstructed visualization during cytological analysis. The use of fluorescent banding tech- niques with CMA and DAPI, has significantly advanced the field of plant cytogenetics by identifying GC- and AT-rich constitutive heterochromatin regions on chro- mosomes, leading to increased knowledge and advance- ments in plant chromosome research (Schweizer 1976; Yamamoto 2012). The current study represents the first documented use of a double staining approach combin- ing CMA and DAPI on chromosomes in T. foenum-grae- cum to date, producing a clear and easily distinguishable banding pattern, marking the first recorded instance of fluorochrome banding in this species based on our cur- rent knowledge. The centromeres, along with secondary constrictions, were reliably designated as CMA+ve and were also correlated with DAPI-ve bands. This establishes that the centromere region has a high concentration of GC nucleotides. A thorough examination of several spe- cies unveiled that the DNA found in centromeres can possess a substantial richness of GC nucleotides. While some animal species exhibit, a predilection for AT-rich tandem repeats, no such tendency was apparent in the plant kingdom (Melters et al. 2013). The detection of CMA+ve centromeric heterochromatin in Crotalaria, a member of the Fabaceae family, implies the existence of GC-rich DNA repeat units at the centromere (Mondin and Aguiar-Perecin 2011). In most species, the rDNA sites exhibit a positive stain, when subjected to CMA staining and a negative stain when treated with DAPI. These sites are frequently the sole regions displaying pos- itive CMA staining (de Melo and Guerra 2003). A com- mon characteristic of plants is the association of GC-rich regions with 35S rDNA sites, resulting in the generation of CMA+ve bands in the NOR (Marcon et al. 2005; Dydak et al. 2009; Kolano et al. 2013). The rDNA sites are gener- ally positively stained with CMA and negatively stained with DAPI. In many species, the rDNA sites are the only regions that are positively stained with CMA. In one pair of chromosomes, positive bands detected through DAPI staining have been identified in the region between the primary and secondary constrictions (Fig. 2b). During prometaphase, when the chromatins are less compact, Figure 2. Karyogram and Idiogram representation of the somatic chromosomes of Trigonella foenum-graecum. (a) Stained with giem- sa; (b) Stained with DAPI; (c) Stained with CMA; (d) Idiogram of the chromosomes along with the localization of different bands. 68 Indranil Santra, Diptesh Biswas, Biswajit Ghosh distinct signals were observed through DAPI staining. This has been documented in several plant species, and the observation that the DAPI signal is only present dur- ing prometaphase and disappears during metaphase suggests that it is not a manifestation of heterochroma- tin, but instead an early stage of chromatin condensa- tion (Berjano et al. 2009; Santra et al. 2021). The use of silver nitrate staining enables the recognition of riboso- mal DNA (rDNA) sites that were transcribing during the preceding interphase of the cell cycle, as visualized in the metaphase stage (Jiménez et al. 1988). In T. foenum-grae- cum, two pairs of chromosomes have been observed with AgNOR bands at secondary constrictions correspond- ing to the CMA+ve bands. In this species, previous stud- ies have documented information about the count and placement of the AgNOR bands, which are in agreement with the results of the current research (Ahmad et al. 1999). The authors also hypothesized that the origin of the two satellite chromosome pairs in fenugreek remains unclear, but it may stem from the hybridization of two distinct species or cytotypes. The localization of AgNOR, CMA, and DAPI bands appear to be valuable cytologi- cal markers, which have enabled us to distinguish and identify the chromosomes in T. foenum-graecum. The standardized techniques of EMA, Giemsa staining, silver staining and fluorochrome banding are considered to be reliable and reproducible. The results of this study hold great significance in understanding the genetic make- up of fenugreek. The study offers critical knowledge on the characterization and preservation of this neglected crop and its diversity, leading to an enrichment of its improvement program. This is vital for maintaining the sustainability of food production and the environment’s well-being. ACKNOWLEDGEMENT Authors are thankful to Swami Kamalasthananda, Principal, Ramakrishna Mission Vivekananda Cente- nary College, Rahara, Kolkata (India), for the facilities provided for the present study. DB acknowledges, Coun- cil of Scientific & Industrial Research (CSIR-HRDG) for providing CSIR – Senior Research Fellowship. REFERENCES Acharya SN, Thomas JE, Basu SK. 2008. Fenugreek, an alternative crop for semiarid regions of North Amer- ica.  Crop Sci  48:841–853. https://doi.org/10.2135/ cropsci2007.09.0519 Agarwal K, Gupta PK. 1983. Cytological studies in the genus Trigonella Linn. Cytologia 48:771–779. 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 Al-Jasass FM, Al-Jasser MS. 2012. Chemical Composition and Fatty Acid Content of Some Spices and Herbs under Saudi Arabia Conditions. Sci World J. 2012:1– 5. https://doi.org/10.1100/2012/859892 Andras SC, Hartman TPV, Alexander J et al. 2000. Com- bined PI–DAPI staining (CPD) reveals NOR asym- metry and facilitates karyotyping of plant chromo- somes. Chromosome Res. 8:387–391. https://doi. org/10.1023/A:1009258719052 Arya ID, Rao SR, Raina SN. 1988. Cytomorphological studies of Trigonella foenum-graecum autotetraploids in three (C1, C2, C3) generation. Cytologia 53:525– 534. https://doi.org/10.1508/cytologia.53.525 Bairiganjan GC, Patnaik SN. 1989. Chromosomal evolu- tion in Fabaceae. Cytologia 54:51–64. Barros e Silva AE, Guerra M. 2010. The meaning of DAPI bands observed after C-banding and FISH proce- dures. Biotech Histochem 85:115–125. https://doi. org/10.3109/10520290903149596 Basu S. 2023. Elucidating karyotype structure and affinity through application of karyomorphological param- eters and multivariate analysis, as discerned from the study of four important legumes. Nucleus 66:39–46. https://doi.org/10.1007/s13237-023-00416-8 Berjano R, Roa F, Talavera S, Guerra M. 2009. Cytotaxon- omy of diploid and polyploid Aristolochia (Aristolochi- aceae) species based on the distribution of CMA/DAPI bands and 5S and 45S rDNA sites. Plant Syst Evol 280:219–227. https://doi.org/10.1007/s00606-009-0184-6 Biddak L. 1996. Inter-and intraspecific chromosomal var- iations in four species of Trigonella L. J Union Arab Biol, Cairo 3:203–215. Bloom SE, Goodpasture C. 1976. An improved tech- nique for selective silver staining of nucleolar organ- izer regions in human chromosomes. Hum Genet 34:199–206. https://doi.org/10.1007/BF00278889 Das AB, Mohanty S, Das P. 2001. Cytophotometric estima- tion of 4C DNA content and karyotype analysis in ten cultivars of Trigonella foenum-graecum. Iran J Bot 9:1–9. Das AB, Mohanty S, Das P. 2002. Cytophotometric esti- mation of 4C DNA content and karyotype analysis in ten cultivars of Trigonella foenum-graecum-II. Iran J Bot 9:151–159. https://doi.org/10.2135/cropsci2007.09.0519 https://doi.org/10.2135/cropsci2007.09.0519 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/10.1100/2012/859892 https://doi.org/10.1023/A https://doi.org/10.1023/A https://doi.org/10.1508/cytologia.53.525 https://doi.org/10.3109/10520290903149596 https://doi.org/10.3109/10520290903149596 https://doi.org/10.1007/s13237-023-00416-8 https://doi.org/10.1007/s00606-009-0184-6 https://doi.org/10.1007/BF00278889 69Chromosomal characterization mediated by karyomorphological analysis and differential banding pattern in fenugreek Das AB, Mohanty S, Thangaraj T, Das P. 2000. Variation of 4C DNA content and karyotype in nine cultivars of fenugreek (Trigonella foenum-graecum L.). J Herbs Spices Med Plants 7:25–32. https://doi.org/10.1300/ J044v07n01_04 de Melo NF, Guerra M. 2003. Variability of the 5S and 45S rDNA sites in Passiflora L. species with distinct base chromosome numbers. Ann Bot 92:309–316. https://doi.org/10.1093/aob/mcg138 de Moraes AP, dos Santos Soares Filho W, Guerra M. 2007. Karyotype diversity and the origin of grape- fruit. Chromosome Res 15:115–121. https://doi. org/10.1007/s10577-006-1101-2 Dydak M, Kolano B, Nowak T, Siwinska D, Maluszynska J. 2009. Cytogenetic studies of three European species of Centaurea L. (Asteraceae). Hereditas 146:152–161. https://doi.org/10.1111/j.1601-5223.2009.02113.x Feyzi S, Varidi M, Zare F, Varidi MJ. 2015. Fenugreek (Trigonella Foenum Graecum) Seed Protein Isolate: Extraction Optimization, Amino Acid Composition, Thermo and Functional Properties. J Sci Food Agric. 95:3165–3176. https://doi.org/10.1002/jsfa.7056 Guerra M. 2000. Patterns of heterochromatin distri- bution in plant chromosomes. Genet Mol Biol. 23:1029–1041. https://doi.org/10.1590/S1415- 47572000000400049 Jahan B, Vahidy AA, Ali SI. 1994. Chromosome numbers in some taxa of Fabaceae mostly native to Pakistan. Ann Mo Bot Gard 792–799. https://doi.org/10.1508/ cytologia.54.51 Jiménez R, Burgos M, de La Guardia RD. 1988. A study of the Ag-staining significance in mitotic NOR’s. Hered- ity 60:125–127. https://doi.org/10.1038/hdy.1988.18 Kar K, Sen S. 1991. A comparative karyological study of root and embryo tissue of a few genera of Legumi- nosae. Cytologia 56:403–408. https://doi.org/10.1508/ cytologia.56.403 Kodama Y, Yoshida MC, Sasaki M. 1980. An improved silver staining technique for nucleolus organ- izer regions by using nylon cloth. Jpn J Hum Genet 25:229–233. https://doi.org/10.1007/BF01997700 Kolano B, Saracka K, Broda-Cnota A, Maluszynska J. 2013. Localization of ribosomal DNA and CMA3/ DAPI heterochromatin in cultivated and wild Ama- ranthus species. Sci Hortic 164:249–255. https://doi. org/10.1016/j.scienta.2013.09.016 Ladizinsky G, Vosa CG. 1986. Karyotype and C-banding in Trigonella section Foenumgraecum (Fabaceae). Plant Syst Evol 153:1–5. https://doi.org/10.1007/ BF00989412 Laxmi V, Gupta MN, Datta SK. 1983. Investigations on an induced green seed coat colour mutant of Trigo- nella foenum-graecum L. Cytologia 48:373–378. htt- ps://doi.org/10.1508/cytologia.48.373 Levan A, Fredga K, Sandberg AA. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52:201–220. https://doi.org/10.1111/j.1601-5223.1964. tb01953.x Levin DA. 2002. The role of chromosomal change in plant evolution. Oxford University Press, New York, USA. Maragheh FP, Janus D, Senderowicz M, Haliloglu K, Kolano, B. 2019. Karyotype analysis of eight cultivat- ed Allium species. J Appl Genet 60:1–11. https://doi. org/10.1007/s13353-018-0474-1 Marcon AB, Barros ICL, Guerra M. 2005. Variation in chromosome numbers, CMA bands and 45S rDNA sites in species of Selaginella (Pteridophyta). Ann Bot 95:271–276. https://doi.org/10.1093/aob/mci022 Martin E, Akan H, Ekici M, Aytac Z. 2011. Karyotype anal- yses of ten sections of Trigonella (Fabaceae). Comp Cytogenet 5:105–121. https://doi.org/10.3897/compcyto- gen.v5i2.969 Melters DP, Bradnam KR, Young HA et al. 2013. Com- parative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evo- lution. Genome Biol 14:1–20. https://doi.org/10.1186/ gb-2013-14-1-r10 Mikić A. 2015. Brief but alarming reminder about the need for reintroducing ‘Greek hay’ (Trigonel- la foenum-graecum L.) in Mediterranean agricul- tures.  Genet Resour Crop Evol 62:951–958. https:// doi.org/10.1007/s10722-015-0260-4 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 Najafi S, Anakhatoon EZ, Birsin MA. 2013. Karyotype Characterisation of Reputed Variety of Fenugreek (Trigonella foenum-graecum) in West Azerbaijan- Iran. J Appl Biol Sci 7:23–26. Petropoulos GA. 2002. Fenugreek: The Genus Trigonella. CRC Press, Boca Raton, Florida, USA. Ranjbar M, Zahra H. 2016. Chromosome numbers and biogeography of the genus Trigonella (Fabaceae). Caryologia 69:223–234. https://doi.org/10.1080/0008 7114.2016.1169090 Rasheed MSAA, Wankhade MV, Saifuddin MSSK, Sudar- shan MAR. 2015. Physico-chemical properties of fenugreek (Trigonella foenum-graceum L.) seeds. Int J Eng Res 4:68–70. Santra I, Halder T, Ghosh B. 2021. Somatic and gamet- ic chromosomal characterization with fluorescence https://doi.org/10.1300/J044v07n01_04 https://doi.org/10.1300/J044v07n01_04 https://doi.org/10.1093/aob/mcg138 https://doi.org/10.1007/s10577-006-1101-2 https://doi.org/10.1007/s10577-006-1101-2 https://doi.org/10.1111/j.1601-5223.2009.02113.x https://doi.org/10.1002/jsfa.7056 https://doi.org/10.1590/S1415-47572000000400049 https://doi.org/10.1590/S1415-47572000000400049 https://doi.org/10.1508/cytologia.54.51 https://doi.org/10.1508/cytologia.54.51 https://doi.org/10.1038/hdy.1988.18 https://doi.org/10.1508/cytologia.56.403 https://doi.org/10.1508/cytologia.56.403 https://doi.org/10.1007/BF01997700 https://doi.org/10.1016/j.scienta.2013.09.016 https://doi.org/10.1016/j.scienta.2013.09.016 https://doi.org/10.1007/BF00989412 https://doi.org/10.1007/BF00989412 https://doi.org/10.1508/cytologia.48.373 https://doi.org/10.1508/cytologia.48.373 https://doi.org/10.1111/j.1601-5223.1964.tb01953.x https://doi.org/10.1111/j.1601-5223.1964.tb01953.x https://doi.org/10.1007/s13353-018-0474-1 https://doi.org/10.1007/s13353-018-0474-1 https://doi.org/10.1093/aob/mci022 https://doi.org/10.3897/compcytogen.v5i2.969 https://doi.org/10.3897/compcytogen.v5i2.969 https://doi.org/10.1186/gb-2013-14-1-r10 https://doi.org/10.1186/gb-2013-14-1-r10 https://doi.org/10.1007/s10722-015-0260-4 https://doi.org/10.1007/s10722-015-0260-4 https://doi.org/10.1139/g11-034 https://doi.org/10.1139/g11-034 https://doi.org/10.1080/00087114.2016.1169090 https://doi.org/10.1080/00087114.2016.1169090 70 Indranil Santra, Diptesh Biswas, Biswajit Ghosh banding of Giloy (Tinospora cordifolia): A berberine synthesizing important medicinal plant of India. Car- yologia. 74:63–73. https://doi.org/10.36253/caryolo- gia-1014 Santra I, Haque SM, Ghosh B. 2020. Giemsa C-banding Karyotype and Detection of Polymorphic Constitu- tive Heterochromatin in Nigella sativa L. Cytologia. 85:85–90. https://doi.org/10.1508/cytologia.85.85 Schweizer D. 1976. Reverse fluorescent chromosome banding with chromomycin and DAPI. Chro- mosoma. 58:307–324. https://doi.org/10.1007/ BF00292840 Shabir PA, Wani AA, Nawchoo IA. 2017. Banding Techniques in Chromosome Analysis. In: Bhat T, Wani A (eds) Chromosome Structure and Aberra- tions. Springer, New Delhi, pp 167–180 https://doi. org/10.1007/978-81-322-3673-3_8 Sharghi H, Azizi M, Moazzeni H. 2020. A karyological study of some endemic Trigonella species (Fabace- ae) in Iran. Caryologia. 73:155–161. https://doi. org/10.13128/caryologia-184 She CW, Jiang XH. 2015. Karyotype analysis of Lablab purpureus (L.) sweet using fluorochrome banding and fluorescence in situ hybridization with rDNA probes. Czech J Genet Plant Breed. 51:110–116. htt- ps://doi.org/10.17221/32/2015-CJGPB Stebbins GL. 1971. Chromosomal Evolution in Higher Plants. Edward Arnold Ltd., London. Syed QA, Rashid Z, Ahmad MH, Shukat R, Ishaq A, Muhammad N, Rahman HUU. 2020. Nutritional and therapeutic properties of fenugreek (Trigonella foenum-graecum): a review. Int J Food Prop. 23:1777– 1791. https://doi.org/10.1080/10942912.2020.1825482 Wani SA, Kumar P. 2018. Fenugreek: A Review on Its Nutraceutical Properties and Utilization in Various Food Products. J Saudi Soc Agri Sci. 17:97–106. htt- ps://doi.org/10.1016/j.jssas.2016.01.007 Yamamoto M. 2012. Recent progress on studies of chro- mosome observation in deciduous fruit trees. J Jpn Soc Hortic Sci. 81:305–313. https://doi.org/10.2503/ jjshs1.81.305 https://doi.org/10.36253/caryologia-1014 https://doi.org/10.36253/caryologia-1014 https://doi.org/10.1508/cytologia.85.85 https://doi.org/10.1007/BF00292840 https://doi.org/10.1007/BF00292840 https://doi.org/10.1007/978-81-322-3673-3_8 https://doi.org/10.1007/978-81-322-3673-3_8 https://doi.org/10.13128/caryologia-184 https://doi.org/10.13128/caryologia-184 https://doi.org/10.17221/32/2015-CJGPB https://doi.org/10.17221/32/2015-CJGPB https://doi.org/10.1080/10942912.2020.1825482 https://doi.org/10.1016/j.jssas.2016.01.007 https://doi.org/10.1016/j.jssas.2016.01.007 https://doi.org/10.2503/jjshs1.81.305 https://doi.org/10.2503/jjshs1.81.305 The chromosome resembles more a crystal than other cell organelles Antonio Lima-De-Faria Karyotype asymmetry in some Scilloideae (Hyacinthaceae) members from Algeria Meryem Nassar1,4,*, Nora Sakhraoui2,4, Gianniantonio Domina3 Chromosome counts and karyotype features of different ecotypes of Allium L. species (Amaryllidaceae – Subg. Melanocrommyum) in Iran Shahla Hosseini*, Hiva Yaghoobi Meiotic behavior and its implications on the reproductive success of Arnebia euchroma (Royle ex Benth.) I.M.Johnst. (Boraginaceae), an important medicinal plant of Trans-Himalaya Irfan Iqbal Sofi1,*, Shivali Verma2, Aijaz H. Ganie1, Namrata Sharma2, Manzoor A. Shah1 Morphological and molecular characterization of Sicilian carob (Ceratonia siliqua L.) accessions Antonio Giovino1, Annalisa Marchese2,*, Fxxxxx Bonanno1, Giovanna Sala2, Francesco Paolo Marra3, Gianniantonio Domina2 Microtubule response to salt stress Emre Köseoğlu, Özlem Aytürk* Chromosomal characterization mediated by karyomorphological analysis and differential banding pattern in fenugreek (Trigonella foenum-graecum L.): a neglected legume Indranil Santra, Diptesh Biswas, Biswajit Ghosh*