Development of Female Gametophyte in Gladiolus italicus Miller (Iridaceae)

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.

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 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 morphologically and anatomically. It is also studied karyologically 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. primulinus, 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 striatum and S. californicum by Lakshmanan and Philip (1971) and Crocus sativus and C. thomasii by Chichiricco (1987Chichiricco ( , 1989.
The aim of this study is to determine the development 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 closely 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 stereomicroscope. 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%, absolute 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, Historesinembedding kit), which was prepared by adding hardener to the basic resin and activator mixture in an appropriate ratio according to manufacturer's protocols (Leica Microsystems, Nussloch). Semi-thin (2 µm) sections taken from the materials embedded in historesin with Leica RM2255 rotary microtome and stained with 1% Toluidine blue (O'Brien et al. 1964). Slides were examined with an Olympus CX31 microscope and photographed by Progress C12 camera.

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 nucellus. 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).

Megagametogenesis
After megasporogenesis, atrophy of the three megaspores 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 mitosis 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 chalazal 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 micropylar 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 apparatus with their walls towards the micropylar side. The filiform 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-  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 differentiates from the tissue in the nucellus between integuments 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.
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 nucellus. When the outer integument initiated, the first meiosis was taking place. A cell wall is formed between the two nuclei after meiosis I. After a short rest period, meiosis 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 degenerated. The degeneration of the three supernumerary meiotic products is hence a case of developmental programmed cell death (PCD) and it is of interest to investigate its features with the aim of checking resemblance to apoptotic morphological syndrome as described in general and as described for plants (Papini et al. 2011). TEM observations on the degenerating nonfunctional megaspores in Larix leptolepis (Sieb. et. Zucc.) Gordon (Pineaceae) 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 supernumerary megaspores in angiosperms is a deletional PCD, since the developmental program leading to the female gametophyte formation and maturation implies their disappearance (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 (Chichiricco 1989), and Iris mandshurica (Zhang et al. 2011).
A large number of tissue remodeling occurs during the seed development, with some of the cells being eliminated as a result of PCD. Synergids die during double 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 louisianica (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, vacuolization 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 secretory 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(Zhang et al. ). Ünal (2011 reported that hypostasis developing from nucellar cells beneath the embryo sac plays a role in preventing 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 degeneration of synergid cells. G. italicus showed characters of the Iridaceae family in terms of female gametophyte development. Data gained from this study will also contribute to the general knowledge about the embryological characters used in the taxonomy of Iridaceae family.