Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(3): 91-97, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1082 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Ciler Kartal, Nuran Ekici, Almina Kargacıoğlu, Hazal Nurcan Ağırman (2021) Development of Female Gametophyte in Gladiolus italicus Mill- er (Iridaceae). Caryologia 74(3): 91-97. doi: 10.36253/caryologia-1082 Received: September 24, 2020 Accepted: July 20, 2021 Published: December 21, 2021 Copyright: © 2021 Ciler Kartal, Nuran Ekici, Almina Kargacıoğlu, Hazal Nur- can Ağırman. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits 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 NE: 0000-0003-2005-7293 ÇK: 0000-0002-8621-7889 Development of Female Gametophyte in Gladiolus italicus Miller (Iridaceae) Ciler Kartal1, Nuran Ekici2,*, Almina Kargacıoğlu1, Hazal Nurcan Ağırman1 1Department of Biology, Faculty of Science, Trakya University, Edirne, 22030, Turkey 2Department of Science Education, Faculty of Education, Trakya University, Edirne, 22030, Turkey *Corresponding author. E-mail : nuranekici@yahoo.com Abstract. In this study gynoecium, megasporogenesis, megagametogenesis and female gametophyte of Gladiolus italicus Miller were examined cytologically and histologically by using light microscopy techniques. Ovules of G. italicus are of anatropous, bitegmic and crassinucellate type. Embryo sac development is of monosporic Polygonum type. Polar nuclei fuse before fertilization to form a secondary nucleus near the antipodals. The female gametophyte development of G. italicus was investigated for the first time with this study. Keywords: Gladiolus italicus, Iridaceae, embryo sac, Polygonum type. INTRODUCTION Iridaceae family is represented by approximately 66 genera and 2245 spe- cies in the world (Christenhusz and Byng 2016; Burgt et al. 2019). This family includes ornamentals like Gladiolus, Belamcanda, Iris, Crocus, Eleutherine, etc. and also plants of commercial value like Crocus sativa and lris spp. (Ven- kateswarlu et al.1980). Genus Gladiolus L. has more than 260 species (Gold- blatt 1996). 11 Gladiolus species are found in various regions of Turkey and 6 of them; G. anatolicus (Boiss.) Stapf, G. attilae Kit Tan, B. Mathew & A. Baytop, G. halophilus Boiss. & Heldr., G. humilis Stapf, G. micranthus Stapf, G. osmaniyensis Sağıroğlu) are endemic (Tan et al. 2006; Güner et al. 2012; Sağıroğlu and Akgül 2014). Gladiolus italicus is in IUCN Red List category of Turkey. G. italicus is distributed in Macaronesia, Mediterranean basin to central Asia. It is also introduced and naturalized in California. It naturally grows in many parts of Turkey (Demir and Çelikel 2019). It is a monocotyle- don with spectacular flowers (Tan and Edmondson 1984). Gladiolus corms are used in the treatment of dysentery and gonorrhea in some countries of Africa (Nguedia et al. 2004). In Turkey, it is known as an aphrodisiac and it is known to have emetic property (Baytop 1999). G. italicus and G. atroviolaceus corms are used in ice cream and also in other 92 Ciler Kartal et al. dairy foods (Öztürk and Özçelik 1991). The chemical composition of Gladiolus plants is studied. Demeshko et al. (2020) studied carboxylic acid content of G. hybridus leaves. The chemical composition of the essential oil and the antibacterial, antifungal and antioxidant properties of the essential oil extract of G. italicus are investigated by Üçüncü et al. (2016). The seed testa structure of G. italicus is studied with scanning electron microscope by Erol et al. (2006). Üzen (1999) studied G. italicus mor- phologically and anatomically. It is also studied karyo- logically and cytologically. The chromosome numbers of the species are resulted 2n=30, 60 (Iran) and 2n=120 (Aegean Islands and Spain) in several populations (Perez and Pastor 1994; Kamari et al. 2001; Fakhraei et al. 2011). In addition, chromosome numbers such as 2n = 60, 90, 110-120, 120, ~170, 176 are reported by other researchers (Ohri and Khoshoo 1985; Van Raamsdonk and De Vries 1989). Mensinkai (1939) reported that cytomixis was observed in meiosis in a study of four Gladiolus species (G. tristis, G. byzantinus, G. primuli- nus, and G. dracoce) mitosis and meiosis divisions. The pollen morphology of G. italicus is examined using light and scanning electron microscopy by Dönmez and Işık (2008). They described the pollen grains of G. italicus as monosulcate, heteropolar, elliptic, spinulose-perforate and tectate-collumellate (Dönmez and Işık 2008). Embryological studies in family Iridaceae are rather limited. Studies about the development of the embryo sac are reported by Davis (1966) in Iris japonica and I. tenax. Then other studies are done in Sisyrinchium stri- atum and S. californicum by Lakshmanan and Philip (1971) and Crocus sativus and C. thomasii by Chichiricco (1987, 1989). The aim of this study is to determine the develop- ment of female gametophyte in G. italicus. Cytological and embryological features of G. italicus have not been studied yet. This study is also an attempt toward a better understanding of taxonomic relationships between close- ly related taxa within the Iridaceae and are indirectly useful to the efforts to protect this species in vitro. MATERIALS AND METHODS In this study, G. italicus plants were collected from Höyüklütatar village of Edirne A1 (E) in European Turkey. They were brought to the Botanical Garden of Trakya University. Voucher specimens were placed in the Herbarium of Trakya University (EDTU). Ovaries were examined under an Olympus SZ61 stereomicro- scope. For cyto-histological studies, flowers and buds were fixed in Carnoy’s fluid (3:1, ethyl alcohol: acetic acid). Dehydration process was done with increasing alcohol series for 24 hours (70%, 80%, 90%, 96%, abso- lute alcohol). Then, the material was kept in a mixture of 1 absolute alcohol: 1 basic resin + activator for 24 hours. After being kept in pure resin for 24 hours, the next day, they were embedded in historesin (Leica, Historesin- embedding kit), which was prepared by adding hardener to the basic resin and activator mixture in an appropri- ate ratio according to manufacturer’s protocols (Leica Microsystems, Nussloch). Semi-thin (2 µm) sections tak- en from the materials embedded in historesin with Leica RM2255 rotary microtome and stained with 1% Tolui- dine blue (O’Brien et al. 1964). Slides were examined with an Olympus CX31 microscope and photographed by Progress C12 camera. RESULTS Gynoecium Gynoecium of  Gladiolus italicus, contains a pis- til  with inferior ovary, a long style and a three-lobed, spatulate stigma (Figure 1). G. italicus has a trilocular, syncarpous ovary. In the ovary 22-24 ovules are margin- al-central placented (Figure 2). Megasporangium Ovules of G. italicus are anatropous, crassinucellate and bitegmic. The outer integument consisted of 5-6 cell layers and the inner integument consisted of 2 cell lines. The micropyle is formed by the inner integument. The inner and outer integuments are five- to seven-layered around the micropyle (Figure 3a). Megasporogenesis One of the sub-epidermal cells at the tip of the ovule of G. italicus differentiates to form a megaspore mother cell (MMC). The MMC cell forms deep within the nucel- lus. It has a large volume and larger nucleus and is easily distinguishable from other cells (Figure 3b). As the outer integument become apparent, the first meiotic division begins in the MMC (Figure 3c). The volume of the MMC increases during meiosis (Figure 3d). A dividing wall is formed between the two nuclei after meiosis I. After a short period, also meiosis II is completed. A linear megaspore tetrad forms as a result of meiosis of the MMC (Figure 3e). 93Megasporogenesis and megagametogenesis in Gladiolus italicus Megagametogenesis After megasporogenesis, atrophy of the three mega- spores on the micropylar side occurred. Then, the active megaspore on the chalazal side began mitosis. When the active megaspore divided into two nuclei at the end of the first mitosis, they moved towards the opposite poles and a large central vacuole was formed between them (Figure 3f). The nuclei in the poles enters in the second mito- sis and an embryo sac with 4 nuclei is formed (Figure 3g). After the third mitosis, there are eight nuclei in the embryo sac. Female gametophyte The embryo sac of G. italicus is of the Polygonum type. It has eight nuclei and seven cells. It shows a clear polarization. The mature embryo sac contains an egg apparatus, three antipodal cells and a central cell with two polar nuclei. The polar nuclei fuse before fertilization and form a secondary nucleus near the antipodal cells. Antipodal cells Antipodal cells are haploid. They have densely stained cytoplasm and evident, large nucleolus (Figure 3h). They are surrounded by whole cell wall. Their chala- zal sides are embedded within the hypostasis. (Figure 3i). Central cell The central cell is located in the middle of the embryo sac, initially contains a polar nucleus on the micropylar side and a polar nucleus near the antipodal cells on the chalazal side. The nucleus on the micropy- lar side moves to the side of the polar nucleus, which is located close to the chalazal side (Figure 3j). These two polar nuclei fuse before fertilization and they form the secondary nucleus near the antipodal cells. (Figure 3k). Egg apparatus The egg apparatus is located on the micropylar side of the embryo sac. It consists of two synergid cells and an egg cell. The synergid cells form the filiform appara- tus with their walls towards the micropylar side. The fili- form apparatus enlarges the wall surface of the synergid cells. This facilitates nutrient and water intake. One of the synergid cells begins to degenerate before fertiliza- Figure 2. Cross section of Gladiolus italicus’ ovary (ov, ovule; ow, ovary wall). Figure 1. Pistil and stamens of Gladiolus italicus. (ov, ovary; sg, stigma; st, stamens; sy, style). 94 Ciler Kartal et al. tion and a vacuole is formed (Figure 3l). The egg cell is located between the two synergid cells in the embryo sac (Figure 3m). Hypostasis In the advanced stages of embryo sac development, tissue differentiation is observed on the chalazal side, in G. italicus. This tissue is named as hypostasis and it dif- ferentiates from the tissue in the nucellus between integ- uments and the chalaza. The hypostasis in the embryo sac of G. italicus consists of cells with thickened walls and it is cup-shaped (Figure 3n). DISCUSSION In this study, the developmental stages of the embryo sac in G. italicus is presented for the first time by using light microscopy techniques. G. italicus shows the characteristics of the Iridaceae family in terms of ovules and embryo sac development (Davis 1966; Lakshmanan and Philip 1971; Chichiricco 1987; 1989; Zhang et al. 2011). 22-24 ovules are located in the inferior, trilocular ovary in G. italicus. They are marginal-central placented. Zhang et al. (2011) reported that Iris mandshurica had also inferior, trilocular ovary, but its ovules were axial placented. Ovules of G. itali- cus are anatropous, bitegminous and crassnucellate like Iris mandshurica. In G. italicus, 5-6 cell lines formes the outer integument and 2 cell lines formes the inner integ- ument. The micropyle is formed by the inner integu- ment in both G. italicus and Iris mandshurica (Zhang et al. 2011). Similar features are observed in Sisyrinchium striatum and S. californicum (Lakshmanan and Philip 1971), Crocus sativus (Chichiricco 1987), C. thomasii Figure 3. Female gametophyte in Gladiolus italicus and its develop- mental stages; 3a, ovule of G. italicus; 3b, Crassinucellate ovule of G. italicus; 3c, Bitegmic ovule of G. italicus; 3d, Prophase I phase of meiosis in megaspore mother cell in G. italicus; 3e, G. italicus’ linear type of megaspore tetrad; 3f, Two-nucleate embryo sac in G. italicus; 3g, Four-nucleate embryo sac in G. italicus; 3h; Antipo- dal cells of G. italicus; 3i, Densely stained cytoplasm and nucleoli in the antipodal cells of G. italicus; 3j, Polar nuclei of G. italicus; 3k, Secondary nucleus of G. italicus; 3l; Synergid cells of G. italicus; 3m, Egg apparatus of G. italicus; 3n, Hypostase in G. italicus. (A, antipodal cell; C, chalaza; dm, degenerated megaspores (arrows); EA, egg apparatus; EC, egg cell; F, funiculus; FA, filiform apparatus; fm, functional megaspore (arrow); H, hypostase; ii, inner integu- ment; MMC, megaspor mother cell; M, micropyle; N, nucleus; Nu, nucleolus; Nuc, Nucellus; oi, outer integument; PN, polar nucleus; S, synergid; SN, secondary nucleus; V, vacuole). 95Megasporogenesis and megagametogenesis in Gladiolus italicus (Chichiricco 1989). In previous studies, it was reported that both outer and inner integuments were formed by 2 cell lines in Sisyrinchium striatum and S. californicum (Lakshmanan and Philip 1971). The inner integument is formed by 2 cell lines in Crocus thomasii (Chichiricco G. 1989). The outer integument is formed by 6-8 layers and the inner integument was formed by 4-6 cell lines in Iris mandshurica (Zhang et al. 2011). These findings showed that G. italicus had characteristics of the Iridaceae fam- ily in terms of ovule type and development. It is also closer to Crocus and Sisyrinchium genera in terms of number of cell lines of the inner integument. One of the subepidermal cells in the ovule of G. italicus differentiates from others to form the megaspore mother cell (MMC). The MMC appears below the nucel- lus. When the outer integument initiated, the first mei- osis was taking place. A cell wall is formed between the two nuclei after meiosis I. After a short rest period, meio- sis II is completed. Linear megaspore tetrad occurs as a result of meiosis. Megasporogenesis is of the Polygonum type. In this kind of embryo sac, in the beginning of megagametogenesis, 3 micropylar megaspores are degen- erated. The degeneration of the three supernumerary meiotic products is hence a case of developmental pro- grammed cell death (PCD) and it is of interest to investi- gate its features with the aim of checking resemblance to apoptotic morphological syndrome as described in gen- eral and as described for plants (Papini et al. 2011). TEM observations on the degenerating nonfunctional mega- spores in Larix leptolepis (Sieb. et. Zucc.) Gordon (Pine- aceae) showed morphological features that are typical of PCD (Cecchi Fiordi et al. 2002). Then, megasporogenesis and PCD in Tillandsia (Bromeliaceae) were studied in a comprehensive study by Papini et al. (2011). The general shrinkage of the cell protoplast and the condensation of the cytoplasm and particularly of the nucleus observed in the degenerating supernumerary megaspores are signs of PCD (Pennell and Lamb 1997). The PCD of the supernu- merary megaspores in angiosperms is a deletional PCD, since the developmental program leading to the female gametophyte formation and maturation implies their dis- appearance (Papini et al., 2011). The chalazal megaspore becomes functional and mature embryo sac is formed after three sequential mitosis. Similar findings have been observed in the previously studied species; Sisyrinchium striatum, S. californicum (Lakshmanan and Philip 1971) Crocus sativus (Chichiricco 1987), C. thomasii (Chichir- icco 1989), and Iris mandshurica (Zhang et al. 2011). A large number of tissue remodeling occurs dur- ing the seed development, with some of the cells being eliminated as a result of PCD. Synergids die during dou- ble fertilization (Doronina et al. 2020) Vacuolization is one of the morphological patterns accompanying PCD in synergids. In Nicotiana tabacum (Tian and Russell, 1997), cytoplasmic vacuolization, in Proboscidea louisi- anica (Mogensen, 1978), Penniseturn glaueum (Chaubal and Reger, 1993), Nicotiana tabacum (Huang and Russel, 1994), Helleborus bocconei (Bartoli et al., 2017) vacuole rupture were seen in synergid cells. In G. italicus, vacu- olization is also occurred in one of the synergids due to early stage of fertilization. In G. italicus, bowl-like hypostasis with thickened walls is seen and it is densely stained. In some plants, although the walls of the hypostasis are thickened due to substances such as cutin, suberin and lignin, in some plants they remain thin walled. They have a secre- tory cell structure (Johri et al. 1992). It is reported that thickened walled hypostasis was seen in Crocus sativus (Chichiricco 1987) and C. thomasii (Chichiricco 1989). In Leucojum aestivum (Amaryllidaceae), hypostasis cells are thin-walled and have abundant cytoplasm (Ekici and Dane 2008). There are no reports on hypostasis in Sisyrinchium striatum, S. californicum (Lakshmanan and Philip 1971) and Iris mandshurica (Zhang et al. 2011). Ünal (2011) reported that hypostasis developing from nucellar cells beneath the embryo sac plays a role in pre- venting embryo growth. They also deliver nutrients from the vascular bundles to the embryo sac. In some taxa they play a role in maintaining the water balance. In the light of these findings, it is seen that G. italicus is close to genus Crocus in terms of hypostasis. CONCLUSION In conclusion, the ovule and the development of female gametophyte of G. italicus were studied for the first time and it was seen that the findings obtained from this study were compatible with the previously examined species belonging to the Iridaceae family. PCD occurred when functional megaspore formed at the end of megasprogenesis. It has also occurred in the degen- eration of synergid cells. G. italicus showed characters of the Iridaceae family in terms of female gametophyte development. Data gained from this study will also con- tribute to the general knowledge about the embryologi- cal characters used in the taxonomy of Iridaceae family. ACKNOWLEDGEMENT This study was supported by Trakya University Sci- entific Research Projects Coordination Unit. Project Number: TUBAP-2019/27 96 Ciler Kartal et al. REFERENCES Bartoli G, Felici C, Castiglione M.R. 2017. Female game- tophyte and embryo development in Helleborus boc- conei Ten. (Ranunculaceae). Protoplasma. 254(1): 491-504. Baytop T. 1999. Türkiye’de Bitkiler ile Tedavi; Geçmişte ve Bugün. Ankara: Nobel Tıp Kitapevi (in Turkish). Burgt XM van der, Konomou G, Haba PM, Magassouba S. 2019. Gladiolus mariae (Iridaceae), a new species from fire-free shrubland in the Kounounkan Massif, Guinea. Willdenowia. 49:117-126. Cecchi Fiordi A, Papini A, Brighigna L. 2002. Pro- grammed cell death of the nonfunctional megaspores in Larix leptolepis (Sieb. et Zucc.) Gordon (Pinaceae): ultrastructural aspects. Phytomorph. 52(2-3):187- 195. Chaubal R, Reger BJ. 1993. Prepollination degeneration in mature synergids of pearl millet: an examination using antimonate fixation to localize calcium. Sex. Plant Reprod. 6(4):225-238. Chichiricco G. 1987. Megasporogenesis and development of embryo sac in Crocus sativus L. Caryologia. 40:59- 69. Chichiricco G. 1989. Embryology of Crocus thomasii (Iri- daceae). Plant Syst. Evol. 168:39-47. Christenhusz MJM, Byng JW. 2016. The number of known plants species in the world and its annual increase. Phytotaxa. 261(3):201-217. Davis G.L. 1966. Systematic embryology of the angio- sperms. New York: Wiley. Demeshko OV, Kovalev VN, Mykhailenko OA, Kriv- oruchko EV. 2020. Carboxylic acids from leaves of Gladiolus hybridus. Chem. Nat. Compd. 56(2):312-314. Demir S, Çelikel FG. 2019. Endangered Gladiolus Species of Turkey. Turkish Journal of Agriculture - Food Sci. Technol. 7(5):693-697. Doronina TV, Sheval EV, Lazareva EM. 2020. Pro- grammed cell death during formation of the embryo sac and seed. Russ. J. Dev. Biol. 51(3):135-147. Dönmez OE, Işık S. 2008. Pollen morphology of Turkish Amaryllidaceae, Ixioliriaceae and Iridaceae. Grana. 47:15-38. Ekici N, Dane F. 2008. Cytological and histological stud- ies on female gametophyte of Leucojum aestivum (Amaryllidaceae). Biologia. 63(1):67-72. Erol O, Uzen E, Kucuker O. 2006. Preliminary SEM observations on the seed testa structure of Gladiolus L. species from Turkey. Int. J. Bot. 2:125-127. Fakhraei LM, Rahimi MA, Ghanavati F. 2011. Karyotyp- ic studies of Gladiolus italicus Mill population. New Cell. Mol. Biotechnol. J. 1(4):37-47. Goldblatt P. 1996. Gladiolus in Tropical Africa: Systemat- ics Biology and Evolution. Portland: Timber Press. Güner A, Aslan S, Ekim T, Vural M, Babaç MT, editors. 2012. Türkiye Bitkileri Listesi (Damarlı Bitkiler). İstanbul: Nezahat Gökyiğit Botanik Bahçesi ve Flora Araştırmaları Derneği Yayını (in Turkish). Huang BQ, Russell SD. 1994. Fertilization in Nicotiana tabacum: cytoskeletal modifications in the embryo sac during synergid degeneration. Planta. 194(2):200- 214. Johri BM, Ambegaokar KB, Srivastava PS. 1992. Compar- ative embryology of angiosperms. Berlin: Springer- Verlag. Kamari G, Blanche C, Garbari F. 2001. Mediterranean chromosome number reports. Flora Mediterr. 11:435- 483. Lakshmanan KK, Philip VJ. 1971. A contribution to the embryology of Iridaceae. Proc. Indian Acad. Sci. 73:110-116. Mensinkai SW. 1939. Cytological studies in the genus Gladiolus. Cytologia. 10:51-58. Mogensen HL. 1978. Pollen tube–synergid interactions in Proboscidea louisianica (Martineaceae). Am. J. Bot. 65(9):953-964. Nguedia JCA, Etoa FX, Benga VP, Lontsi D, Kuete Y, Moyou RS. 2004. Anti-candidal property and acute- toxicity of Gladiolus gregasius Baker (Iridaceae). Pharma. Méd. Tradi. Africa. 13:149-159. O’Brien TP, Feder N, McCully ME. 1964. Polychromatic staining of plant cell walls by Toluidine Blue O. Pro- toplasma. 59:368-373. Ohri D, Khoshoo TN. 1985. Cytogenetics of garden Gladiolus II. Variation in chromosome complement and meiotic system. Cytologia. 50:213-231. Öztürk M, Özçelik H. 1991. Useful plants of East Anato- lia. Siirt: Siirt İlim, Spor, Kültür ve Araştırma Vakfı Yayını. Papini A, Mosti S, Milocani E, Tani G, Di Falco P, Brighi- gna L. 2011. Megasporogenesis and programmed cell death in Tillandsia (Bromeliaceae). Protoplas- ma. 248:651–662. Pennell RI, Lamb C. 1997. Programmed cell death in plants. Plant Cell, 9:1157-1168. Perez E, Pastor J. 1994. Contribution al studio cariolog- ico de la familia Iridaceae en Andalucia occidental. Lagascalia. 17:257-272. Sağıroğlu M, Akgül G. 2014. Gladiolus osmaniyensis (Iri- daceae), a new species from South Anatolia, Turkey. Turk. J. Botany. 38:31-36. Tan K, Edmondson JR. 1984. Gladiolus L. In: Davis PH, editor. Flora of Turkey and the East Aegean Islands, Vol. 8. Edinburgh: Edinburgh University Press. 97Megasporogenesis and megagametogenesis in Gladiolus italicus Tan K, Mathew B, Baytop A. 2006. Gladiolus attilae (Iri- daceae), a new species from East Anatolia, Turkey. Phyt. Balc. 12:71-73. Tian HQ, Russell SD. 1997. Calcium distribution in fer- tilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L. Planta. 202(1):93–105. Üçüncü O, Baltacı C, İlter SM. 2016. Chemical com- position and bioactive properties of the volatile oil of Gladiolus italicus Miller. Gümüşhane Univ. J. Sci. Technol. Inst. 6:150-156. Ünal M. 2011. Bitki (Angiosperm) Embriyolojisi. İstanbul: Nobel Yayın Dağıtım (in Turkish). Üzen E. 1999. Türkiye’nin bazı Gladiolus (Iridaceae) tür- leri üzerinde biyosistematik araştırmalar. PhD the- sis, İstanbul Üniversitesi Fen Bilimleri Enstitüsü, İstanbul. Van Raamsdonk LWD, De Vries T. 1989. Biosystematic studies in European species of Gladiolus (Iridaceae). Plant Syst. Evol. 165:189-198. Venkateswarlu J, Sarojini DP, Nirmala A. 1980. Embry- ological studies in Eleutherine plieata Herb. and Belamcanda ehinensis Lem. Proc. Indian Acad. Sci. 89(5):361-367. Zhang D, Wang L, Zhuo L. 2011. Embryology of Iris mandshurica Maxim. (Iridaceae) and its systematic relation. Plant Syst. Evol. 293:43-52. 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