Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(2): 31-39, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2124 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Biplab Kumar Bhowmick, Sayani Nag (2023). Unfolding chromo- somal uniqueness of the Scilloid orna- mental Albuca virens by application of EMA based Giemsa- DAPI staining. Caryologia 76(2): 31-39. doi: 10.36253/ caryologia-2124 Received: April 25, 2023 Accepted: October 19, 2023 Published: December 31, 2023 Copyright: © 2023 Biplab Kumar Bhow- mick, Sayani Nag. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided 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 BKB: 0000-0001-6029-1098 SN: 0009-0009-1353-892X Unfolding chromosomal uniqueness of the Scilloid ornamental Albuca virens by application of EMA based Giemsa- DAPI staining Biplab Kumar Bhowmick*, Sayani Nag Department of Botany, Scottish Church College, 1&3, Urquhart Square, Kolkata- 700006, West Bengal, India *Corresponding author. E-mail: biplab.bhowmick@scottishchurch.ac.in Abstract. The cytogenetic features in the tribe Ornithogaleae of subfamily Scilloideae is a prerequisite for understanding genome evolution. Unfortunately, genomic or the foundational chromosomal features are neglected within majority of Ornithogaloids, incuding Albuca, one of the largest genus of the tribe. Albuca virens (Lindl.) J.C. Man- ning & Goldblatt is the only ornamental species of Albuca found to be exotic to India. Analysis of karyotype by EMA method followed by Giemsa and DAPI staining is the first step towards molecular cytogenetics attempted in this species and has currently brought significant resolution in chromosome morphology (especially NORs). The Indian population of A. virens with 2n=6 chromosomes, symmetric karyotype and heterophorphy of NORs provide excellent scope to navigate questions on dysploid origin of Albuca. The regular meiotic stages advocate genomic stability despite veg- etative propagation and polysomaty in root cells. The comparative review of chromo- somal evolution within Albuca has been discussed in relation to the Indian A. virens as a prototype. Keywords: Albuca virens, EMA, DAPI, meiosis, dysploidy, NORs. INTRODUCTION The genus Albuca belongs to the tribe Ornithogaleae of Asparagaceae/ Hyacinthaceae, subfamily Scilloideae, sensu APG III 2009; APG IV 2016) with more than 100 species distributed mainly in sub-Saharan Africa (Gold- blatt and Manning 2011; Martinez- Azorin et al. 2011). Considering repeated taxonomic amendments and changes in species circumscription (Martinez- Azorin 2011, 2023; Manning 2020), chromosomal features are given special attention to resolve species boundaries within the tribes of Asparagaceae/ Hyacinthaceae (Goldblatt and Manning 2011). It is evident from the recent compilation that cytogenetic investigation is so far confined to chromo- some counts or karyotypes by conventional staining methods in less than 50% of accepted species belonging to tribes Hyacintheae, Urgineeae, Orni- 32 Biplab Kumar Bhowmick, Sayani Nag thogaleae and Oziroëeae (Nath et al. 2022). Albuca virens (Lindl.) J.C. Manning & Goldblatt of subgenus Urophyllon (Salisb.) J.C. Manning & Goldblatt (Man- ning et al. 2009a,b) has been found to occur as an orna- mental exotic to India while some wild populations are reported from north eastern part (Bhattacharya et al. 2016). Within subgenus Urophyllon of Albuca, karyotype analysis by fluorochrome staining is reported in Albuca bracteata (Thunb.) J.C.Manning & Goldblatt (worked out as syn. Ornithogalum longibracteatum Jacq.), the sister species of A. virens (Pedrosa et al. 2001). Nomenclatural ambiguity in previous cytological reports is another rea- son for blurring analysis of chromosomal relationships in Albuca. A. virens has four subspecies of which A. virens ssp. virens is previously known and worked out as different species of Ornithogalum with varying chromo- some counts viz. Ornithogalum virens Lindl. (2n=6), O. flavovirens Baker (2n=10), O. ecklonii Schldl. (2n=10), O. tenuifolium Delaroche (2n=12, 10, 8, 16, 6, 4), O. preto- riense Baker (2n=12) or O. inconspicuum Baker (2n=20) (Goldblatt and Manning 2011). In our country also, very few traditional cytological studies have been conduct- ed on A. virens, in the name of the syn. Ornithogalum virens (Ravindran 1977; Bhattacharya et al. 2016). Owing to the backdrops of conventional cytological studies, lucid karyotype features of A. virens is still missing. Hence the aim of the present work is to obtain bet- ter cytogenetic analysis with enzymatic maceration and air drying (EMA) method (Kurata and Omura 1978; Fukui 1996) in A. virens over the traditional orcein staining approach. In spite of being a well-known alter- nate method for chromosome analysis (Fukui and Iijima 1991; Yamamoto et al. 2010, 2015; Nath et al. 2015; Jha et al. 2020; Jha 2021; Bhowmick and Jha 2022), EMA based approach has not been yet attempted in Albuca virens. Our adoption of EMA method enabled lucid karyotype interpretation, fluorescence staining and understand- ing meiotic chromosome behavior in this plant. The outcome of the present paper upgraded existing knowl- edge about chromosome configuration in this species and provides foundational data for taxonomic revisions, genome research and hybridization breeding of A. virens as a potential ornamental plant. MATERIALS AND METHODS Plant materials Bulbs of Albuca virens were collected from local nurseries of Darjeeling district of West Bengal, India as ornamentals and were duly identified by Dr. Manoj M. Lekhak, Shivaji University, Kolhapur, India. The bulbs are potted, grown and maintained in the medici- nal plant garden of Scottish Church College, Kolkata (Figs. 1a, b). Actively growing underground roots were used for chromosome analysis. Young flower buds from inf lorescence (Fig. 1b) were picked to study meiotic chromosomes. Chromosome preparation Pre-treatment of healthy underground secondary roots (0.5- 1 cm in length) was done in 0.002M hydrox- yquinoline at 15 °C for 4 hours and then roots were fixed in freshly prepared 1:3 aceto-methanol solution. Mitotic frequency and chromosome morphology were firstly studied by conventional squashing method. Fixed roots were then subjected to enzymatic maceration and air drying method (Kurata and Omura 1978; Fukui 1996) after necessary standardization (Jha and Bhowmick 2021; Bhowmick and Jha 2022) for the present plant material. Roots were firstly digested in a cocktail of 1% cellulase (Onuzuka RS), 0.15% pectolyase (Y-23), 0.75% macerozyme (R-10) and 1mM EDTA (pH 4.2) for vary- ing time durations (45-50 min) at 37 °C before macera- tion on clean glass slides in a drop of 1:3 aceto-methanol solution. Slides with macerated roots were air-dried and stained with 2% Giemsa solution (Giemsa azure eosine methylene blue solution Merck Germany: 1/15th phos- phate buffer: distilled water :: 2:50:48) for 15 min. Met- aphase plates were observed under 100X objectives of Zeiss Axioscope microscope with attached Axiocam 202 mono camera and Zen software for capturing photomi- crographs. Karyotype preparation and analysis Minimally ten different metaphase plates from seven individual plants were selected for chromosome measurements [long arm length (l), short arm length (s), chromosome length (CL), total diploid chromatin length (TCL= )]. Chromosomes were categorized after Levan et al. (1964) on the basis of r value (l/s) and arranged in an order of decreasing length for construct- ing karyotypes and ideograms. Any trend of asymme- try in karyotype was determined after calculating val- ues of twelve different indices. These include Stebbins asymmetry index (Stebbins 1971), total form percentage (TF%) (Huziwara 1962), intrachromosomal asymme- try index (A1) and interchromosomal asymmetry index (A2) (Zarco 1986), coefficients of variation of chromo- some length (CVCL) (Paszko 2006), index of karyotype symmetry (Syi) (Greilhuber and Speta 1976), asymme- 33Detailed karyotype analysis of Albuca virens try index of karyotype (AsK%) (Arano 1963), degree of karyotype asymmetry (A) (Watanabe et al. 1999), mean centromeric index (XCI) (Seijo and Fernández 2003), dispersion index (DI) (Lavania and Srivastava 1992), asymmetry index (AI) (Paszko 2006) and mean cen- tromeric asymmetry (MCA) (Peruzzi and Eroğlu 2013). Considering the absence of the allied Albuca species in India, chromosome morphometric data have been bor- rowed from a published report involving Ornithogalum comosum Sadler, O. montanum Cirillo, O. pyrenaicum L. and O. sigmoideum Freyn & Sint. (Öztürk et al. 2014) to compare the asymmetry indices and comment on trend of karyotype evolution in Albuca virens. Since only chro- mosome lengths and centromeric indices were provided in the published paper (Öztürk et al. 2014), only A2, CVCL, XCI and AI values could be estimated for the refer- ence taxa. Fluorochrome staining of somatic metaphase chromosomes The Giemsa stained slides were marked under the f luorescent microscope Zeiss Axioscop5 followed by destaining in 70% methanol for 45min. Fluorochorme staining with DAPI (4’,6-diamidino-2-phenylindole) was performed following Schweizer (1976) after required standardization and modifications (Bhowmick and Jha 2021; Jha and Bhowmick 2021). Slides were incubated in McIlvaine buffer (0.1M citric acid, 0.2M Na2HPO4, pH 7.0) for 30 min and stained with 0.1mg/ml DAPI solu- tion for 25-30 min in dark. Excess stain from slides was washed off in McIlvaine buffer followed by blow drying and mounting in non-fluorescent glycerol. Observation of DAPI stained metaphase plates was carried out under the Zeiss Axioscop2 with UV filter cassette. Fluorescent chromosome images were captured with the attached Axiocam 202 mono camera and Zen software. Meiotic chromosome preparation following DAPI staining Young f lower buds of approximately 0.5-0.8cm length were collected at around 10a.m. Initial screening of the meiotic stages were conducted by staining PMCs (pollen mother cells) in 2% acetocarmine solution. Due to dense cytoplasmic content in the PMCs, EMA- DAPI staining (Bhowmick and Jha 2015) was conducted with minor modifications. The anthers were isolated from flower buds and then digested in enzyme cocktail (same as mentioned for somatic chromosome preparation) for 2-4mins at 37°C. Macerated anthers were pipetted out in clean glass slides and P.M.C.s were spread in 1:3 v/v acetic acid- ethanol solution and air dried. The slides Figure 1. a. Whole plant. b Part of inflorescence with flowers, floral organs, fruit and seeds in insets. c-e Somatic metaphase chromo- somes (2n=6) stained with orcein (c, d) and polysomatic cell pho- tograph stained with Feulgen (2n=12) (e), note obscure chromo- some morphology in conventional staining. f-n Somatic metaphase chromosomes (2n=6) represented from three individual plants: f, i, l Giemsa stained chromosomes with corresponding hand draw- ings (g, j, m) and subsequent DAPI stained plates (h, k, n). Arrows indicate satellite part of chromosomes in Giemsa plates that are cor- respondingly DAPI-ve. o-q Somatic chromosomes showing polyso- matic condition (2n= 12) stained with Giemsa (o), DAPI (p-q). 34 Biplab Kumar Bhowmick, Sayani Nag were kept in McIlvaine buffer (0.1M citric acid, 0.2M Na2HPO4, pH 7.0) for 10min. Slides were then stained with 0.1mg/ml DAPI solution for 20min in dark. Excess stain was washed off in Mc Ilvaine’s buffer and slides were mounted in non-fluorescent glycerol. Slides were observed and images of meiotic stages were captured under UV filter cassette of Zeiss Axioscope 5 fluores- cence microscope with attached Axiocam 202 mono camera and Zen software. RESULTS AND DISCUSSIONS Somatic chromosome morphology and karyotype In the conventional method, cytoplasmic density was difficult to overcome and visualization of chromo- some morphology was problematic (Fig. 1c-e). Applica- tion of EMA method enabled elimination of cytoplasmic background and rendered chromosome spreading in one plane with clear morphology (Fig. 1f-q) which was not possible in previous reports due to limitations of con- ventional method (Ravindran 1977; Bhattacharya et al. 2016). There are six chromosomes in the diploid comple- ment (2n=6) (Fig. 1c, d, f-n), with few records of 2n=12 chromosomes in some cells (4-8%), corresponding with previous reports (Bhattacharya et al. 2016) (Fig. 1e, o-q). Present study shows that the chromosomes are telocen- tric in nature with one satellited pair of chromosomes. The chromosomes range in size from 6.88 to 10.83μm (Table 1), with an average TCL (total length of diploid complement) of 53.20±14.58. The modal karyotype of A. virens is 4t+2T.sat (Fig. 2a-c), showing no signs of distinct difference of short or long chromosomes and hence Indi- an population of A. virens does not show bimodal nature of karyotype (Table 1). However, every pair has at least a little difference in chromosome length between the hom- ologues (Table 1). The largest chromosomes are the ones with a terminal satellite (Table 1). Centromeric position in this pair cannot be determined as they are terminal- ly located, only the satellite part is visible in metaphase plates (Fig. 1f-n, 2a-c). Again, the size of the satellited region varies between the homologues (Fig. 1f-n). In the previous works, 2n=6 was consistently associ- ated with O. virens and 2n=4, 6, 12, 16, 26 was reported in the African A. virens (worked out as Ornithogalum tenuifolium) (Goldblatt and Manning 2011). However, Ornithogalum tenuifolium was included later in Stellari- oides (Castiglione and Cremonini 2012). Other counts (2n=8, 10 and 12) were reported in O. setifolium, O. eck- Figure 2. a-c Idiogram and Giemsa (b)- DAPI (c) karyotype. d-g Carmine stained meiotic chromosomes from PMCs at metaphase II (e) with three chromosomes in each cell of diad, anaphase II showing regular segregation (d, f, g), telophase II indicated with arrow (f), showing no signs of abnormality. h-l DAPI stained meiotic chromosomes from PMCs at diakinesis (h-j) and metaphase I (k, l) showing 3 bivalents. 35Detailed karyotype analysis of Albuca virens lonii, O. flavovirens and O. pretoriense, respectively, all of these species being now identified as Albuca virens (Goldblatt and Manning 2011). Again, one population of A. virens from southern Mozambique shows 2n=4 (Stedje 1989), with one long metacentric pair and one medium sized sub telocentric pair. So far, the ancestral base number of x=10 is suggested for Albuca, with many evidences for derived base numbers x=9 (for subgenera Albuca, Monarchos, Osmyne) and x=6 (subgen. Urophyl- lon) (Goldblatt and Manning 2011). Later, 2n=6 was sug- gested to be a primitive cytotype that may have poten- tially led to origin of 2n=4 by chromosome translocation and fusion (Castiglione and Cremonini 2012). The other phylogenetically sister species with 2n= 18, 54 (A. brac- teata, syn O. caudatum, O. longibracteatum), are suggest- ed to have x=9 (Goldblatt and Manning 2011). Extensive cytological interpretations led Goldblatt and Manning (2011) conclude dysploidy to be a characteristic of sub- genus Urophyllon, with particularly extreme intraspecific dysploidy notable in Albuca virens. Previously, bimodal karyotype has been reported in diploid populations of A. virens, like its sister species Albuca volubilis from Mad- agascar (Goldblatt and Manning 2011). The species of subgenus Albuca consistently show bimodal karyotypes (Knudtzon and Stedje 1986; Jong 1991; Stedje 1996; John- son 1999). Although, the present population of A. virens in India does not have bimodal karyotype, differences in chromosome length between the homologues may be expressions of ongoing intraspecific rearrangements, as reported for cytotypes with 2n=6 (Castiglione and Cre- monini 2012). Considering the difference in the size of satellites in the present study, heteromorphy in the size of the satellite NORs is reported in sections of subgenus Albuca (Jong 1991) and hence can be treated as a con- served phenomenon in A. virens of subgenus Urophyllon. Knowledge about the symmetric/asymmetric nature of karyotype is a fundamental requirement to gain substantial concept of evolution in any group (Liang and Chen 2015). The inter-chromosomal asymme- try indices depict heterogeneity in chromosome sizes while intra-chromosomal asymmetry depends on rela- tive centromere position (Paszko 2006). Unfortunately, detailed chromosome measurement data of Albuca spe- cies is missing in literature (Ravindran 1977; Knudtzon and Stedje 1986; Jong 1991; Stedje 1996; Johnson 1999; Pedrosa 2001). Therefore, the present karyomorpho- metric analysis has been conducted in comparison with some species of Ornithogalum by retrieving a few chro- mosome morphometric records from published data (Öztürk et al. 2014) (Table 2). According to the values determined, A. virens shows symmetric nature of kar- yotype, considering decreased values for A2, CVCL, XCI and AI (Table 3). Symmetric nature of karyotype is justi- fied also according to Stebbins’ index (2A) (Table 3). The chromosomes do not have discrete difference in size or centromeric position, in contrast to the bimodal karyo- types of other Albuca species and the Ornithogalum taxa referred in the present analysis (Table 3). The present detail of karyotype asymmetry parameters can be used as a reference to the allied species of Albuca in future studies. Confirmation of nucleolar chromosomes following DAPI staining The same Giemsa stained chromosome plates were subjected to DAPI staining. Chromosomes were brightly stained with DAPI except at the short arm which was relatively faintly stained (Fig. 1f-n). Comparing the same metaphase plate stained with Giemsa and then with DAPI, satellite part of the two chromosomes was found to be the DAPI-ve NORs (Fig. 1h, k, n). Thus, DAPI stain- ing supported confirmation of NORs in the somatic metaphase chromosomes. DAPI-ve NORs are a common Table 1. Chromosome morphometric features of Albuca virens. Chromosome no. Long arm (l) (μm) Short arm (s) (μm) Sat (μm) Chromosome length (μm) r=l/s Type RL 1 8.5 ± 2.18 0.68± 0.08 - 9.18±2.34 12.25 ± 2.51 t 17.37 ± 0.75 2 7.87± 2.66 0.788± 0.28 - 8.65± 2.80 10.52 ±4.36 t 15.99± 1.18 3 7.718± 2.66 0.75± 0.11 - 8.47± 2.63 10.57±4.44 t 15.91± 2.67 4 6.15± 1.90 0.73± 0.18 - 6.88± 1.98 8.67±2.98 t 12.92± 0.73 5 8.65±2.27 - 2.18± 1.14 10.83± 3.14 - T. sat 20.35± 1.67 6 7.48±1.52 - 1.8±1.30 9.28± 2.61 - T. sat 17.43± 0.45 The data are shown as average values± standard deviation from ten different metaphase plates chosen from seven individual plants. Sat: satellite part of chromosome, r: arm ration (Levan et al. 1964), RL: relative length calculated as: Chromosome length/ Total Chromatin Length*100. 36 Biplab Kumar Bhowmick, Sayani Nag feature of plant chromosomes since nucleolar regions are usually enriched with GC- heterochromatin and thus take up dense stain after application of GC- spe- cific fluorochrome namely chromomycin A3 (Schweizer 1976; Guerra et al. 2000). Albuca virens also conforms to the usual negative banding pattern of DAPI at NORs. Previously, DAPI+ve signals on centromeric and inter- calary regions and CMA+ve/DAPI-ve signals on nucleolar regions are reported in the phylogenetically close A. brac- teata (Pedrosa et al. 2001). Therefore, distribution of non- nucleolar AT-rich heterochromatin (depicted by DAPI+ve bands, Bhowmick and Jha 2015, 2019, 2021) may follow differential distribution among the species of subgenus Urophyllon, to be confirmed after analysis of DAPI band- ing pattern in A. volubilis and related species in question (Goldblatt and Manning 2011). Till then, DAPI-ve NORs remain to be a conserved chromosomal landmark so far in the subgen. Urophyllon of Albuca. In future, chromo- Table 2. Chromosome morphometric data from Ornithogalum species retrieved from published work (Öztürk et al. 2014). Ornithogalum comosum: Ornithogalum montanum: Ornithogalum pyrenaicum : Ornithogalum sigmoideum: Chromosome Pair no. ChL (A) (µm) CI (A) Chromosome Pair no. ChL (A) (µm) CI (A) Chromosome Pair no. ChL (A) (µm) CI (A) Chromosome Pair no. ChL (A) (µm) CI (A) 1 7.84 19.87 1 4.64 36.45 1 10.5 43.75 1 7.8 32.63 2 6.58 27.35 2 4.2 30.39 2 7.28 30.12 2 6.64 27.2 3 6.05 22.72 3 3.68 39.92 3 7.05 21.71 3 6.51 24.31 4 5.86 24.81 4 3.31 33.34 4 6.78 27.06 4 6.22 41.08 5 5.55 35.36 5 3 20.48 5 6.28 27.46 5 5.7 46.28 6 5.04 23.65 6 2.88 22.52 6 5.96 22.75 6 5.2 26.16 7 4.46 22.6 7 1.84 34.98 7 4.46 44.26 7 4.2 31.71 8 4.35 24.02 8 4.39 40.85 9 4.12 27.12 9 3.48 28.16 10 3.08 45.39 10 2.91 36.74 11 2.68 37.15 12 1.6 40.86 ChL: chromosome length, CI: centromere index, A: average. Table 3. Karyotype Symmetry/Asymmetry values in Albuca virens estimated with quantitative and qualitative indices in comparison with published data from Ornithogalum species (Öztürk et al. 2014). Quantitative parameters Qualitaive parameter Inter- Chromosomal Symmetry/ Asymmetry Intra-Chromosomal Symmetry/Asymmetry Inter- & Intra- (Combined) Chromosomal Asymmetry Stebbins asymmetry index A2 CVCL Syi TF% Ask% A1 A XCI MCA DI AI Albuca virens 0.14 14.48 6.38 5.55 86.98 0.06 0.74 1.50 74.66 8.31 2.04 2A Ornithogalum comosum* 0.25 25.98 - - - - - 2.72 - - 7.24 - O. montanum* 0.27 27.45 - - - - - 4.45 - - 6.36 - O. pyrenaicum* 0.47 47.32 - - - - - 2.78 - - 11.52 - O. sigmoideum* 0.19 19.02 - - - - - 4.98 - - 4.73 - A2: Interchromosomal asymmetry index (Romero-Zarco 1986); CVCL: Coefficient of variation of chromosome length (Paszko 2006); Syi: Index of karyotype symmetry (Greilhuber and Spelta 1976 ); TF%: Total form percentage (Huziwara 1962); AsK%: Asymmetry index of kar- yotype (Arano 1963); A1: Intrachromosomal asymmetry index (Romero-Zarco 1986) ; A: Degree of karyotype asymmetry (Watanabe et al 1999 ); XCI : Mean centromeric index (Seijo et al. 2003) ; MCA: Mean centromeric asymmetry (Peruzzi and Eroglu 2013) ; DI: The dispersion index (Lavania and Srivastava 1992); AI: Asymmetry index (Paszko 2006); Stebbins asymmetry index A-C,1-4 (Stebbins 1971);* data derived from previously published average chromosome length and average centromeric index of four Ornithogalum species (Öztürk et al. 2014). 37Detailed karyotype analysis of Albuca virens mycin A3+ve banding or FISH with nucleolar rDNA probe in A. virens and related species is needed to complement our observation. Additionally, somatic paring is reported in A. bracteata around the AT rich intercalary hetero- chromatin which was also claimed long back in A. virens (Ravindran 1977). However, in the present population, neither AT rich intercalary DAPI+ve bands nor such asso- ciation trend was noted. Meiotic chromosome study Different stages of meiosis were obtained following carmine staining, showing regular chromosome segre- gation patterns (Fig. 2d-g). After EMA method followed by DAPI staining, clearly visible bivalents were observed in diakinesis and metaphase I of PMCs (Fig. 2h-l). The haploid number was confirmed as n=3. The absence of any earlier gametic count reports, haploid number of A. virens was questionable, especially considering the occurrence of polysomaty (Bhattacharya et al. 2016, pre- sent study). Presently, regular chromosome paring (Fig. 2h-l) and segregation (Fig. 2d-g) is confirmed in Albuca virens, in line with successful fruit and seed set in spite of frequent vegetative propagation. This information is a pre-requisite for infra- or inter-specific crosses for flori- culture purposes as in the Ornithogalum species (Gries- bach et al. 1990, 1993). CONCLUSION The karyotype of Albuca virens shows distinguish- able features like i) diploid number 2n=6, ii) variation in length of chromosomes between homologues and iii) variation in the size of NORs. With the knowledge of dysploid reduction and several rearrangements among the chromosomes, the Indian A. virens certainly stands as one unique population of Albuca, like the one found by Stedje (1989) from Mozambique with 2n=4. It is ques- tionable whether occurrence of polysomatic numbers in the present plants represents evidences in support of an unstable chromosomal background or a usual feature associated with bulbous ornamentals enjoying abun- dant vegetative propagating (Sharma and Sharma 1956). Considering the literatures in support of dysploidy and chromosome evolution from 2n=6 (Castiglione and Cre- monini 2012), present study adds to the significance of this species as a prototype to analyze chromosome evo- lution with the help of modern cytogenetic methods. In course of the conventional mitotic chromosome analy- sis, it was felt that the chromosomes remain in differ- ent planes, rendering analysis of morphology difficult, as we also encounter inadequate karyotype information in earlier report of A. virens (Bhattacharya et al. 2016). Successful preparation of EMA-based Giemsa- DAPI stained slides and DAPI staining of PMCs open the path for application of molecular cytogenetics like florescence in situ hybridization or Ag-NOR staining. Therefore the present dataset along with the technical standardization part, would be an asset for molecular cytogenetic assess- ment of evolution of the understudied Albuca virens in relation to its allied species. ACKNOWLEDGEMENTS BKB sincerely thanks Principal, Scottish Church College for providing the Scottish Church College Fac- ulty Research Grant (SCCFRG) 2021-2023 for conduct- ing the present research work. BKB thanks Prof. Sumi- ta Jha, Department of Botany, University of Calcutta, India, for kindly gifting the bulbs of Albuca virens. BKB thanks Dr. Manoj M. Lekhak, Angiosperm Taxonomy Laboratory, Department of Botany, Shivaji University, Kolhapur, India, for identification of the plant specimen. 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Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 76, Issue 2 - 2023 Firenze University Press Molecular classification of Barbeyaceae (Barbeya oleoides Schweinf.) using four different DNA barcodes Fatima Omari Alzahrani, Sami Asir Al-Robai Mitotic metaphase karyotype of the mosquito Anopheles arabiensis Patton (Diptera: Culicidae) from Kassala State, eastern Sudan Asma Mahmoud Hamza1,*, Sumaya Hussein Elboshra2 Karyotypic analysis of Crucian carp, Carassius carassius (Linnaeus, 1758) from cold waters of Kashmir Himalayas Gousia Jan1, Asim Iqbal Bazaz2, Azra Shah1, Saima Andleeb1, Irfan Ahmad1,*, Durdana Qazi1, Oyas Asimi3, Bilal A. 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