Microsporogenesis, microgametogenesis and in vitro pollen germination in the endangered species Fritillaria stribrnyi (Liliaceae)

Abstract

Fritillaria stribrnyi Velen. (Liliaceae) is an endangered plant that naturally grows only in southern Bulgaria and Turkey in Europe. In this study, microsporogenesis, microgametogenesis, in vitro pollen germination and spermatogenesis were investigated in F. stribrnyi using a light microscope. Young flower buds and mature pollen grains, collected from natural habitats (in Edirne, Turkey), were used as materials. Microsporogenesis was investigated by the usual acetic-orcein squash method. Microgametogenesis slides were prepared with the paraffin method. Pollen fertility was estimated by lactophenol-aniline blue staining. For pollen germination, pollen grains were collected by immediately dehiscing anthers and then incubating in basic germination medium at 25°C for 24 h. Generally, the phases of meiosis were regular, although there were a few abnormalities such as laggard chromosomes in meiosis I and asynchronization in phases of meiosis II. The meiotic division resulted in the formation of isobilateral types of tetrads. The first mitotic division in pollen was observed clearly, and generative and vegetative cells were formed regularly. The germination percentage of pollen grains was found to be 84%, and the lengths of tubes were measured as 1896.22 ± 760.50 μm. F. stribrnyi has an elegant flower, and, therefore, its cultivation is possible in the garden as an ornamental plant. This is the first embryological study of F. stribrnyi, and the results of this study of the reproduction biology of this endangered plant will be useful for in vitro, in situ and ex situ conservation and genetic studies.

Keywords:

• Fritillaria stribrnyi
• Liliaceae
• endangered
• microsporogenesis
• microgametogenesis
• in vitro pollen germination

Introduction

Liliaceae is one of the larger families of flowering plants, containing about 250 genera and approximately 3500 species. The family is represented by 35 genera and 461 species in the Flora of Turkey (Davis 1984; Davis et al. 1988; Güner et al. 2000). The family has genera that are ornamental plants such as Fritillaria, Lilium and Tulipa.

Fritillaria L. genus is represented in northern temperate regions by seven subgenera and 165 taxa. The genus has 35 species (41 taxa) in Turkey, 21 species of which are endemic (Tekşen and Aytaç 2011). It contains 25 taxa in Greece, six species in Bulgaria, and 22 taxa in Russia (Tomovic et al. 2007; Tekşen and Aytaç 2008). Many species of the genus, which are wild growing, are cultivated for their attractive flowers. Bulbs of some species have been exported from Turkey for over 100 years (Ulug et al. 2010). Turkey is very rich with respect to both the total number of species and endemic species of the genus because the Mediterranean region is a center of diversity for the genus Fritillaria (Tomovic et al. 2007).

Fritillaria stribrnyi Velen. grows in a limited region of southern Bulgaria and northwest Turkey. This species is distributed naturally only in European Turkey (Edirne, Kırklareli, Istanbul and Canakkale) (Rix 1984; Başak 1991). The conservation status of the species, according to the Turkish Red Data Book, is “Endangered” (EN) (Ekim et al. 2000) in Turkey, while it is “Critically Endangered” in Bulgaria, according to Petrova and Vladimirov (2009).

Studies related to the Fritillaria genera that grow in Turkey are very limited. They are usually taxonomical, caryological, morphological and anatomical studies (Başak 1991; Bozbek 2007; Özler and Pehlivan 2007; Tekşen and Aytaç 2008; Tekşen et al. 2010; Tekşen and Aytaç 2011). Recently, micropropagations of F. imperialis and F. persica, which are economically important and ornamental species, were made via plant tissue culture methods (Dilik 2006; Ulug et al. 2010). There are a few taxonomical, caryological and palinological papers on F. stribrnyi (Başak 1991; Tekşen and Aytaç 2008; Tekşen et al. 2010). The species has 2n = 24 chromosomes (Başak 1991) and monosulcate pollen grains (Tekşen et al. 2010). As a result of the literature survey, embryological studies on Fritillaria tenella, F. persica, F. imperialis, F. messanensis, F. pudica, F. japonica, F. ussuriensis and F. liliacea species have been performed (Davis 1966; Noda 1975; Yu-fen and Jia-heng 1990). However, there are no data on the embryology of F. stribrnyi.

The species is represented by a low number of small populations in Turkey, and it is facing a very high risk of extinction caused by human pressures in the wild. The habitat of the species is generally under Paliurus spina-christi in meadows and pastures. Their habitats are rapidly declining due to the destruction of pastures. This work is part of an ongoing project on the reproduction biology and conservation biology of F. stribrnyi. The aim of the study is to provide a comprehensive description of the meiosis and pollen mitosis in anther loculi, in vitro pollen germination in liquid germination medium, and spermatogenesis (mitosis II) in germinated pollen tubes of F. stribrnyi, which is an endangered species.

Material and methods

Flower buds and pollen grains of Fritillaria stribrnyi were used as materials. Materials were collected from the natural habitat in pastures of Üyüklütatar village in Edirne Province (European Turkey).

Microsporogenesis

Flower buds of different sizes were taken from healthy plants at 10:00–10:30AM in February–April 2011, and they were immediately fixed in the field in 3:1 (v/v) ethanol:glacial acetic acid solution. The buds were incubated in fixative at room temperature for 24 h, and then they were transferred to 70% ethanol and stored in a refrigerator until use. For meiosis stages, anthers of different sizes were squashed in a drop of acetic orcein after hydrolysis in 1 N HCl for 10 min at 60°C. Prepared slides were stored in a freezer at –18°C. Cover slips were removed from frozen slides. To make permanent slides, both the slides and cover slips were placed first in ethyl alcohol and then in xylene prior to mounting with entellan.

Pollen mitosis I

The anthers were dissected from fixed flower buds and dehydrated by a gradually increasing ethyl alcohol series. They were then embedded in paraffin (58°C) after the process of paraffin infiltration in xylene (Jensen 1962). Paraffin-embedded samples were sectioned by a Leica rotary microtome (RM 2255) (Leica Biosystems, Nussloch, Germany) in a thickness of 10 μm, and sections were stained Alcian blue (pH 2.5) – Nuclear fast red (K.M. Lyons Lab/UCLA; https://www.mcdb.ucla.edu/Research/Lyons/Protocols_files/ Alcianblue_NFR_paraffin_lyonslab.PDF). The slides were dehydrated by a gradually increasing ethyl alcohol series and, then they were passed through an ethyl alcohol-xylene series and transferred to xylene prior to mounting with entellan.

Pollen viability

The pollen grains were collected from flowers soon after the dehiscence of anthers. For pollen viability tests, pollen grains were stained with a drop of lactophenol-aniline blue solution for 30 min at room temperature. Stained pollen grains were considered fertile, and unstained pollen grains were considered sterile. A total of 3000 pollen grains, both stained and unstained, were counted in 10 randomly chosen regions, and pollen viability was calculated.

In vitro pollen germination

Anthers of F. stribrnyi were collected one day before anthesis and were placed in Petri dishes at room temperature for 24 h. After a day, the pollen from the dehisced anthers was put in test tubes containing 1 ml of liquid germinating medium consisting of 10% sucrose, 0.01% H₃BO₃, 0.03% Ca(NO₃)₂.4H₂O, 0.02% MgSO₄.4H₂O and 0.01% KNO₃ for 24 h at 25°C. Samples were then fixed with an acid-alcohol mixture (1 glacial acetic acid: 3 ethyl alcohol) following 24 h inoculation. The germinating pollens were transferred to slides and stained with acetic-orcein or lactophenol-aniline blue. Slides were mounted in glycerin-gelatin to make permanent preparations (Jensen 1962). Measuring 30 randomly selected zones on slides for 3000 pollen grains determined the in vitro germination percentages. The length of pollen tubes was measured on in vitro germinated pollen grains. Pollen was considered germinated when the pollen tube was equal to or longer than the diameter of the pollen grains. Pollen tube lengths were measured using Image-Pro Plus, version 5.1 (Media Cybernetics 2005). For tube length measurements, normal pollen tubes on 30 randomly selected fields from 10 slides were considered. The experiment was repeated three times.

Spermatogenesis in germinated pollen tubes

The division of the generative cell in germinated pollen tubes was examined on slides stained with acetic-orcein. All observations were carried out using an Olympus BH2 light microscope (Tokyo, Japan), and images were captured by a ProgRes C12 digital camera (Jenoptik, Jena, Germany).

Results

F. stribrnyi is a rare, wild plant that has elegant flowers and is facing a very high risk of extinction in the wild. Microsporogenesis, microgametogenesis, pollen viability, in vitro pollen germination and spermatogenesis within the pollen tube are described in detail in the following sections.

Microsporogenesis and pollen mitosis I

The microspore mother cells (MMCs) (Figure 1a) undergo two consecutive meiotic divisions and form an isobilateral microspore tetrad. Each MMC passes through prophase I (leptotene, zygotene, pachytene, diplotene and diakinesis, shown respectively in Figure 1b–f), metaphase I (Figure 1g), anaphase I (Figure 1h) and telophase I (Figure 1i). The cytokinesis that occurs is the successive type, and the dyads are formed at the end of meiosis I (Figure 1j). Callose begins to accumulate around the microsporocytes from the leptotene stage. Meiosis II in each dyad via prophase II (Figure 1k), metaphase II (Figure 1l), anaphase II (Figure 1m) and telophase II (Figure 1n) results in isobilateral tetrads (Figure 1o). The phases of meiosis in F. stribrnyi are generally regular, but there are a few abnormalities in 3% of cases such as lagging chromosomes in metaphase I (Figure 2a) and telophase I (Figure 2b) and asynchrony in the stages of meiosis II (Figure 2c, d).

Figure 1. (Color online) Microsporogenesis in Fritillaria stribrnyi stained with acetic-orcein. (a) Microspore mother cells; (b) leptotene; (c) zygotene; (d) pachytene; (e) diplotene; (f) diakinesis; (g) metaphase I; (h) anaphase I; (i) telophase I; (j) dyad; (k) prophase II; (l) metaphase II; (m) anaphase II; (n) telophase II; (o) isobilateral tetrad. Scale bars = 20 μm.

Figure 2. (Color online) Abnormalities in Fritillaria stribrnyi during microsporogenesis. (a) Lagging chromosome in metaphase I (arrow); (b) lagging chromosome in telophase I (arrow); (c, d) asynchrony in the stages of meiosis II. Scale bars = 20 μm.

When the microspores just release from the tetrad, they have dense cytoplasm and centrally placed and irregularly shaped nuclei (Figure 3a). Later, the exine develops, and the germination aperture occurs on the uni-nucleate microspore (Figure 3b). Many small vacuoles appear in the cytoplasm. They fuse into a large, central vacuole and push the cytoplasm together with the nucleus against the microspore margin (Figure 3c). The nucleus is located in a position opposite the aperture. Then, young pollen grains undergo pollen mitosis I (prophase, Figure 3d), and the mitotic spindle forms asymmetrically at the metaphase stage (Figure 3e). Following the anaphase (Figure 3f) and the telophase (Figure 3g) stages, a curved phragmoplast forms between nuclei (Figure 3h), and the cytokinesis occurs asymmetrically. Finally, a generative cell with dense chromatin and smaller cytoplasm and a vegetative cell with loose chromatin and larger cytoplasm form (Figure 3i). Pollen grains are 2-celled when released from the anther.

Figure 3. (Color online) Microgametogenesis in Fritillaria stribrnyi.(a) Microspore just released from the tetrad; (b) microspore with developed exine and aperture; (c) vacuolated microspore; (d) prophase; (e) metaphase; (f) anaphase; (g) telophase; (h) phragmoplast formation at the end of telophase (arrow); (i) vegetative and generative cells in pollen grain (arrow shows the wall of generative cell) (gc, generative cell; v, vacuole; vc, vegetative cell). Scale bars = 20 μm.

Pollen viability and in vitro pollen germination

The fertile pollen grains of F. stribrnyi are well-stained with lactophenol aniline blue solution, while the sterile pollen grains are shriveled and empty, and they are not stained (Figure 4). The percentage of fertile pollen grains is 92.72%.

Figure 4. (Color online) Fertile and sterile pollen grains of Fritillaria stribrnyi stained with lactophenol-aniline blue solution (fp, fertile pollen; sp, sterile pollen). Scale bar = 50 μm.

In vitro pollen germination observations are performed on germinated pollen grains that are stained with aceto-orcein or lactophenol-aniline blue following incubation at 25°C for 24 h. Pollen grains begin to germinate rapidly after contact with the liquid germination medium. After 10 min, the pollen tube is over twice the size of the pollen grain in equatorial diameter. The germination rate of pollen grains is calculated as 84%, and the pollen tube length is measured as 1896.22 ± 760.50 μm after 24 h incubation (Figure 5). The vegetative nucleus enters first into the pollen tube and then the generative nucleus enters into the observed pollen tubes (Figure 6). Pollen tubes are generally regular, but some pollen tubes produce swollen or branched tube tips (Figure 7a, b). The rate of these abnormalities is 4%.

Figure 5. (Color online) Pollen tubes germinated at 25°C for 24 h and stained with lactophenol-aniline blue solution. Scale bar = 200 μm.

Figure 6. (Color online) Generative and vegetative nuclei stained with acetic-orcein in germinated pollen tube (gn, generative nucleus; vn, vegetative nucleus). Scale bar = 50 μm.

Figure 7. (Color online) Pollen tube abnormalities of Fritillaria stribrnyi. (a) Swollen pollen tube tip; (b) branched pollen tube tips. Scale bars = 20 μm.

Spermatogenesis in germinated pollen tubes

Spermatogenesis (pollen mitosis II) always occurs in pollen tubes after tube formation in in vitro germination medium. While the generative cell in bicellular mature pollen grains is ellipsoid-shaped (Figure 3i), it is long and cylindrical-shaped with blunt ends in the pollen tube (Figure 6). In the pollen tubes, which are fixed and stained after 24 h, all the stages of mitosis are easily observed. At prophase, the chromatin begins to condense in the nucleus (Figure 8a), then the nuclear envelope disappears and the chromosomes lie in the generative cell cytoplasm (Figure 8b). At metaphase, a typical equatorial plate forms, and the chromosomes arrange at the plate (Figure 8c). During anaphase, the sister chromatids regularly separate and move toward opposite poles as individual chromosomes in the tube (Figure 8d). At telophase, the chromosomes begin to uncoil, and the nuclear envelope reforms around each chromosome group (Figure 8e, f). Eventually, two sperm nuclei occur inside the pollen tube (Figure 8g). Although spermatogenesis is generally regular, chromosome bridges of different thicknesses are seen in some tubes at telophase (1%) (Figure 8h, i).

Figure 8. (Color online) Spermatogenesis of Fritillaria stribrnyi in in vitro germinated pollen tube. (a) Prophase; (b) prometaphase; (c) metaphase; (d) anaphase; (e) telophase; (f) the end of telophase; (g) two sperm nuclei; (h) telophase bridge (arrow); (i) telophase bridge (arrow) Scale bars: (a–h) 20 μm; (i) 10 μm.

Discussion

This study reports microsporogenesis, pollen mitosis I, pollen viability, in vitro pollen germination and spermatogenesis within pollen tubes of F. stribrnyi, which is facing a very high risk of extinction caused by human pressures in the wild. For successful sexual reproduction in plants, microsporogenesis, microgametogenesis and pollen germination is very important. Therefore, these events have been at the center of interest for botanists.

The phases of meiosis in F. stribrnyi are generally regular, but there are a few abnormalities in 3% of cases such as the lagging chromosomes in the stages of meiosis I and asynchrony in the stages of meiosis II. Similar abnormalities are seen in Gagea stipitata (Liliaceae) (Koul et al. 1976), Gagea lutea (Bohdanowicz et al. 2005), Tulipa clusiana (Liliaceae) (Wafai and Koul 1982), Bellevalia edirnensis (Hyacinthaceae) (Dane 2006), Leucojum aestivum (Amaryllidaceae) (Ekici and Dane 2012) and Allium senescens (Liliaceae) (Zhang et al. 2012). These abnormalities can be caused by genetic or environmental factors. Bohdanowicz et al. (2005) report that the dividing microsporocytes of Gagea lutea are affected by cold treatment (4°C) and drying in a laboratory, and these conditions increase abnormalities in the stages of microsporogenesis as compared to cold and drying in natural conditions. Shiva Kameshwari and Muniyamma (2001) explain that there are more meiotic disturbances of the tetraploid species Chlorophytum elatum (2n = 28) (Liliaceae) than the diploid species C. heynei and C. laxum (2n = 14). Cytokinesis of meiosis in F. stribrnyi is successive, and the tetrads show isobilateral (or tetragonal) arrangement such as in most of the monocotyledons and the other Liliaceae members (Davis 1966; Yu-fen and Jia-heng 1990; Furness and Rudall 1999). However, tetrahedral tetrads together with isobilateral tetrads are reported in Tulipa clusiana (Wafai and Koul 1982) and Gagea stipitata (Koul et al. 1976). In three species of Chlorophtum (Liliaceae) – C. elatum, C. heynei and C. laxum – only isobilateral tetrads were seen by Shiva Kameshwari and Muniyamma (2001).

Asymmetric cell division, which is dependent on the formation of a typical hemispherical phragmoplast, is very important for normal differentiation of the vegetative and generative cells in pollen mitosis I. The free microspores released from tetrads in F. stribrnyi divide asymmetrically to give rise to a large vegetative cell and a small generative cell. Before the division, the cytoplasm becomes vacuolated, and the microspore nucleus migrates towards the edge of the pollen. According to Ekici and Dane (2004), this region, to which the nucleus migrates during pollen mitosis, determines the location of generative cells and is controlled genetically. These events are similarly seen in the other monocotyledones such as Tulipa clusiana (Liliaceae) (Wafai and Koul 1982), Oryza sativa (Poaceae) (Dane and Meric 2005), Bellevalia edirnensis (Hyacinthaceae) (Dane 1999), Agapanthus praecox (Agapanthaceae) (Zhang et al. 2010) and Leucojum aestivum (Amaryllidaceae) (Ekici and Dane 2012).

The disturbances in the course of microsporogenesis in F. stribrnyi are few, and thus the pollen fertility rate is high (92.72%). Li et al. (2003) suggest that the abnormalities in meiosis are the major cause of abortive pollen in Lilium davidii var. unicolor (Liliaceae). As in most Liliaceae members (Davis 1966), including Fritillaria ussuriensis (Yu-fen and Jia-heng 1990), mature pollen grains are two-celled when released from the anther of F. stribrnyi. In Allium senescens (Liliaceae), a few three-celled pollen grains are found along with the usual two-celled, as observed by Zhang et al. (2012).

The pollen grains of F. stribrnyi begin to germinate rapidly in liquid medium. The germination rate is very high at the end of 24 hours (84%). It is has been shown that the content of the germination medium is very important for F. stribrnyi. There are many studies related to in vitro pollen germination of Liliaceae members in solid or liquid media. The germination rate in fertile genotypes of Allium sativum is about 80% in solid medium containing 15% sucrose (Shemesh Mayer et al. 2013). The pollen germination rate of Ornithogalum caudatum (Liliaceae) is the greatest in 10% sucrose, and no germination is observed in 30% sucrose concentration (Goss 1962). The in vitro germination rates of Asparagus species (Liliaceae) is the greatest in 10% sucrose concentration for diploid A. racemosus and 30% for tetraploid A. densiflorus (Dhoran et al. 2011). Dhoran et al. (2011) report that the genotype has an important role in in vitro pollen germination.

The average tube length is 1896.22 ± 760.50 μm after 24 h incubation. The pollen tubes of F. stribrnyi can grow about 79 μm per hour within 24 h. Aksoy et al. (2013) report that the average pollen tube length in Amsonia orientalis is 1678 μm after 12 h in the germination medium. The tube length of germinating pollen is measured as approximately 95.25 ± 38 μm in Hypericum perforatum and 165.92 ± 53 μm in Hypericum rumeliacum 4 h after inoculation (Arda et al. 2006).

The spermatogenesis of F. stribrnyi is easily observed in germinated pollen tubes after 24 hours. At the end of prophase, the nuclear membrane disappears, and the chromosomes are arranged in metaphase plate. Anaphase and telophase stages are generally regular, although there are a few chromosome bridges (1%). Two sperm nuclei that are surrounded by a little cytoplasm occur at the end of telophase. There are many papers on generative cell division of the Liliaceae members, such as some Lilium (Cooper 1936) and Tulipa species (Upcott 1936; Johnston 1941), Gagea lutea (Zhang et al. 1995), Hyacinthus orientalis (Del Casino et al. 1992), Ornithogalum virens (Banas et al. 1996) and Convallaria majalis (Del Casino et al. 1999). Liliaceae members exhibit different properties related to the formation of the metaphase plate during the division of the generative cell. Lilium (Cooper 1936) and Tulipa species (Upcott 1936; Johnston 1941), Gagea lutea (Zhang et al. 1995) and Ornithogalum virens (Banas et al. 1996) have a distinct metaphase plate, while in Hyacinthus orientalis (Del Casino et al. 1992) and Convallaria majalis (Del Casino et al., 1999), a typical metaphase plate seems lacking. For Fritillaria genus, this paper is the first report on generative cell division in growing pollen tubes.

The present study aims to explain the reproduction biology of F. stribrnyi, an endangered species which is facing a very high risk of extinction caused by human pressures in the wild, and the results of this study provide useful data for further in vitro, in situ and ex situ conservation programs for this species.

Acknowledgment

This study was supported by The Scientific Research Fund of Trakya University, Turkey [Project No: TUBAP – 2009 / 100].