Meiotic Defects and Premature Tapetal Degeneration Are Involved in the Low Fertility of Oncidesa Gower Ramsey, an Important Cut-flower Orchid

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  • 1 Pingtung Seed and Seedling Research Center, Taiwan Seed Improvement and Propagation Station, Council of Agriculture, Executive Yuan, 90943 Pingtung, Taiwan, R.O.C.
  • | 2 Department of Horticulture and Landscape Architecture, National Taiwan University, 10617 Taipei, Taiwan, R.O.C.
  • | 3 Biology Department, National Museum of Natural Science, 40453 Taichung, Taiwan, R.O.C.; and Department of Life Sciences, National Chung Hsing University, 40227 Taichung, Taiwan, R.O.C.

The dancing-lady orchid, Oncidesa Gower Ramsey, is an important cultivar for cut-flower production, but it has low pollen fertility in breeding programs. In this study, we compared the pollen germination in vitro, sporad type, and pollinia development of Oncsa. Gower Ramsey and a diploid species, Oncidium sphacelatum Lindl (one of its grandparents). In Oncsa. Gower Ramsey, the pollen germination in vitro was lower as compared with those in Onc. sphacelatum. In addition, the frequency of abnormal sporads in Oncsa. Gower Ramsey was higher than those in Onc. sphacelatum. In Oncsa. Gower Ramsey, the middle layer and the tapetum were disorganized before meiosis, and subsequently they degenerated at the early tetrad stage. In contrast, the middle layer and the tapetum of Onc. sphacelatum began to degenerate at the early tetrad stage and fully disappeared at the bicellular pollen stage. These results suggested that the abnormal meiosis caused by unbalanced genomes and the premature degeneration of the middle layer and the tapetum could probably result in the abnormal pollen development and the low fertility of Oncsa. Gower Ramsey.

Abstract

The dancing-lady orchid, Oncidesa Gower Ramsey, is an important cultivar for cut-flower production, but it has low pollen fertility in breeding programs. In this study, we compared the pollen germination in vitro, sporad type, and pollinia development of Oncsa. Gower Ramsey and a diploid species, Oncidium sphacelatum Lindl (one of its grandparents). In Oncsa. Gower Ramsey, the pollen germination in vitro was lower as compared with those in Onc. sphacelatum. In addition, the frequency of abnormal sporads in Oncsa. Gower Ramsey was higher than those in Onc. sphacelatum. In Oncsa. Gower Ramsey, the middle layer and the tapetum were disorganized before meiosis, and subsequently they degenerated at the early tetrad stage. In contrast, the middle layer and the tapetum of Onc. sphacelatum began to degenerate at the early tetrad stage and fully disappeared at the bicellular pollen stage. These results suggested that the abnormal meiosis caused by unbalanced genomes and the premature degeneration of the middle layer and the tapetum could probably result in the abnormal pollen development and the low fertility of Oncsa. Gower Ramsey.

The dancing-lady orchid, Oncidium Sw., and its relative genera within subtribe Oncidiinae native to Central and South America are important ornamental orchids in the horticultural market (Chase, 1997). Because of the long-lasting and bright yellow flowers, the cultivars of intergeneric hybrid Oncidesa (Oncidium × Gomesa R. Br.), for example, Oncsa. Gower Ramsey and Oncsa. Goldiana, are excellent cut-flower materials for decoration in flower arrangements. In Taiwan, Oncsa. Gower Ramsey has been an important cultivar for cut-flower production over the past few decades. The creation of new cultivars to meet the requirements of the floral market is essential for maintaining a competitive advantage. In the breeding program of Oncidium alliance, hybridization allows the transfer of novel traits from one cultivar to another with the chances of selecting superior cultivars (Hieber et al., 2006; Moir and Moir, 1982). Although Oncsa. Gower Ramsey is a good cultivar for cut-flower production, the low percentage of capsule-set is commonly observed when Oncsa. Gower Ramsey is used as the pollen parent in our previous pollination experiments.

The causes of low fertility may be associated with abnormal meiosis in orchid hybrids. Meiotic irregularities, such as univalents, trivalents, chromosome bridges, and lagging chromosomes, are observed commonly in the interspecific or intergeneric hybrids of Aranda (Lee, 1987), Dendrobium Lindl. (Kamemoto et al., 1999), Doritaenopsis (Bolanos-Villegas et al., 2008), and Paphiopedilum Pfitz. (Lee et al., 2011), and the irregular meiotic pairing subsequently causes the formation of abnormal sporad types. In addition, it is possible that male sterility or low fertility is related to the malfunction of anther tissue. The sporophytic anther tissues play important roles in pollen development, not only by providing physical support but also by supplying signals and materials necessary for pollen development. Especially, the tapetum, the most inner somatic cell layer of the anther locule, plays an essential role in pollen development. Tapetum secrets enzymes for the release of microspores from tetrads and provides nutrients and pollen wall materials required for pollen development and maturation (Goldberg et al., 1993; Hegde and Rudramuniyappa, 1986; Wilson and Yang, 2004). It has been observed that the abnormal development or dysfunction of tapetum may lead to the pollen degeneration in a number of plants, for example, Arabidopsis thaliana (L.) Heynh (Kim et al., 2010), garlic (Shemesh-Mayer et al., 2015), kiwifruit (Giuseppina et al., 2013), tomato (Jeong et al., 2014), Solanum Lindl. (Conicella et al., 1997), snap bean (Suzuki et al., 2001), petunia (Kapoor et al., 2002), radish (Shi et al., 2010), and rice (Ku et al., 2003; Shi et al., 2009).

The pollinia development of orchids has been reported in Calanthe Lindl. (Hegde and Rudramuniyappa, 1986), Epidendrum Lindl. (Yeung, 1987; Yeung and Blackman, 1983), Zeuxine Lindl. (Vijayaraghavan et al., 1987), Peristylus Lindl. (Zee and Siu, 1990), Aranda (Lee, 1987), and some intergeneric hybrids, for example, Cattleya Lindl., Laelia Lindl., and Brassavola Lindl. (Stort, 1984). However, limited information is available about the pollinia development of Oncidesa, and there are no reports about the possible roles of anther tissues in relation to the male sterility or low fertility. Histological study on pollinia development is required to provide the necessary background information before embarking on breeding programs. To get a better understanding of the low fertility in Oncsa. Gower Ramsey as the pollen parent, we compared the pollinia development of Oncsa. Gower Ramsey and its diploid parental species, Onc. sphacelatum (with good fertility). In addition, their sporad types after meiosis, pollen viability, and capsule setting by cross-pollination also were examined. The present study provides detailed information of the pollinia development at the light microscope level and extends our knowledge concerning pollen development in Oncidesa.

Materials and Methods

Plant material.

Mature plants of Onc. sphacelatum and Oncsa. Gower Ramsey were cultivated in the greenhouse of Botanical Garden of National Museum of Natural Science, Taiwan. Plants were grown in pine bark medium (Orchiata; Besgrow, Christchurch, New Zealand) under 50% shade. Plants were fertigated monthly with a 20N–20P–20K Peters Professional 20–20–20 general purpose fertilizer (Scotts, Marysville, OH) at 0.5 g·L−1. For histological and sporad type studies, developing flower buds of different stages were collected at 3-d intervals until anthesis. More than 100 developing flower buds and flowers were collected for this study.

Light microscopy.

Developing flower buds were collected and fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) buffered with 0.1 m sodium phosphate buffer, pH 6.8, at 4 °C overnight. After three 15-min buffer rinses, the materials were postfixed in 1% OsO4 (Electron Microscopy Sciences) in the same buffer for 4 h at room temperature, then rinsed in three 15 min changes of buffer. Following fixation, the materials were dehydrated in an acetone series (Merck KGaA, Darmstadt, Germany), and embedded in Spurr’s resin (Electron Microscopy Sciences). Serial sections 1 μm thick were cut, using a diamond knife, on an ultramicrotome (Ultracut E; Reichert-Jung, Vienna, Austria). These sections were stained with 0.1% (w/v) toluidine blue O in benzoate buffer for general histology. The sections were viewed and the images were captured digitally using a charge-coupled device camera attached to a light microscope (Axioskop 2; Carl Zeiss AG, Jena, Germany).

Sporad type observation.

At anthesis, the pollinia were collected and fixed in fresh prepared Farmer’s fluid (three parts of ethanol to one part of glacial acetic acid). The pollinia were macerated with 6% cellulose (Onozuka R-10; Yakult Honsha, Japan) and 6% pectinase (Sigma-Aldrich Co., St. Louis, MO) in 75 mm KCl, pH 4.0, at 37 °C for 90 min and squashed on slides with the same fixative. After drying, the slides were stained with 4′,6-diamidino-2-phenylindole in VECTASHIELD (Vector Laboratories Inc., Burlingame, CA), and the images were captured digitally by a charge-coupled device camera attached to an epifluorescence microscope (Axioskop 2; Carl Zeiss AG).

In vitro pollen germination.

At the time of anthesis, pollinia were collected from the flowers of Onc. sphacelatum and Oncsa. Gower Ramsey. After removing anther caps, the surface of pollinia was wiped with cotton soaked with 70% ethanol solution. In the laminar flow hood, six pollinia mass were transferred to one petri dish containing 15 mL of germination medium. The germination medium used in this study was the modified Brewbaker and Kwack medium (Brewbaker and Kwack, 1963), containing 20 g·L−1 sucrose, 100 mg·L−1 H3BO3, 200 mg·L−1 MgSO4‧7H2O, 300 mg·L−1 Ca(NO3)2‧4H2O, 100 mg·L−1 KNO3, and solidified with 10 g·L−1 agar (Sigma-Aldrich Co.). The pH value was adjusted to 5.5 with 1 N NaOH solution before autoclaving at 121 °C for 15 min. The cultures were maintained at 25 ± 1 °C in the dark chamber, and the pollinia were taken out for observation at 1, 2, 4, 6, 8, and 10 d of culture. The germinating pollinia were transferred onto slides and gently separated by forceps, then were stained with 0.04% methyl green (Sigma-Aldrich Co.) for pollen tube observation under a light microscopy. Germination of pollen was defined as emergence of the pollen tube from the pollen grain. The germination percentage was evaluated as the percentage of the number of pollen grains germinated among the total countered number of pollen grains. Experiments were performed in a randomized design.

Capsule setting by cross-pollination.

For cross-pollination experiments, Oncsa. Gower Ramsey was cross-pollinated with Onc. sphacelatum or Gom. Java, respectively. The reciprocal crosses were made at the same time. The flowers were labeled and manually cross-pollinated by transferring pollinia from the male parent onto the stigma of the female parent as soon as they opened. The capsule setting was calculated 2 months after cross-pollination.

Results

The pollen germination in vitro.

In Onc. sphacelatum, the pollen tube began to grow by 2 d after culture, whereas in Oncsa. Gower Ramsey, the pollen tube began to grow by 6 d after culture. By 10 d after culture, the percentage of pollen germination in vitro in Onc. sphacelatum was much greater (68.29 ± 5.21%) as compared with those in Oncsa. Gower Ramsey (2.30 ± 1.39%) (Table 3).

The pollen development.

In Onc. sphacelatum and Oncsa. Gower Ramsey, at the early sporogenous stage, the sporogenous cells had nuclei of great volume with conspicuous nucleoli and the dense cytoplasm (Fig. 1A and B). The primary parietal layer underwent periclinal divisions, giving rise to multilayered microsporangial walls that differentiated into the endothecium, the middle layer, and the tapetum (Fig. 1C and D). The sporogenous cells gave rise to form microspore mother cells (MMCs) that were filled with numerous small vacuoles (Fig. 1C and D). The endothecial cells continued to enlarge by the process of vacuolation, whereas the cells of the middle layer and the tapetum remained cytoplasmic. Before meiosis, in Onc. sphacelatum, the tapetal cells were elongated and possessed prominent nuclei (Fig. 1E). In MMCs, the chromatins further condensed in preparation for meiotic divisions (Fig. 1E). On the contrary, in Oncsa. Gower Ramsey, the cells of the middle layer and the tapetum were disorganized with the appearance of condensed nuclei (Fig. 1F). In Onc. sphacelatum and Oncsa. Gower Ramsey, MMCs underwent meiotic divisions and resulted in the formation of microspore tetrads (Fig. 2A and B). In Onc. sphacelatum, the middle layer and the tapetum began to degenerate after meiosis (Fig. 2A). In Onc. sphacelatum and Oncsa. Gower Ramsey, haploid microspores were not released from tetrads after meiosis, and all tetrads aggregated to form the pollinium. The microspore within a tetrad underwent pollen mitosis I (Fig. 2A), giving rise to a vegetative cell and a generative cell (Fig. 2C and D). At anthesis, the mature pollen tetrads within a pollinium of Onc. sphacelatum contained numerous lipid bodies and protein bodies (Fig. 2E), whereas in Oncsa. Gower Ramsey, a number of mature pollen tetrads shrank and degenerated (Fig. 2F).

Fig. 1.
Fig. 1.

Micrographs of the microspore mother cell (MMC) development of Onc. sphacelatum and Oncsa. Gower Ramsey. In (A) Onc. sphacelatum and (B) Oncsa. Gower Ramsey, their sporogenous cells (S) have dense cytoplasms with nuclei of great volume. Cell divisions in the primary parietal layer give rise to multilayered microsporangial walls that differentiate into the endothecium (Ed), the middle layer (M), and the tapetum (T) in (C) Onc. sphacelatum and (D) Oncsa. Gower Ramsey, and the sporogenous cells give rise to form MMCs. In Onc. sphacelatum (E), the chromatins of MMCs further condense in preparation for meiotic divisions, and the tapetal cells have elongated and possess prominent nuclei, whereas in Oncsa. Gower Ramsey (F), the cells of the middle layer and the tapetum are disorganized with the appearance of condensed nuclei. Scale bar = 10 μm.

Citation: HortScience horts 53, 9; 10.21273/HORTSCI13011-18

Fig. 2.
Fig. 2.

Micrographs of the microgametogenesis of Onc. sphacelatum and Oncsa. Gower Ramsey. Meiotic divisions (arrowhead) result in the formation of microspore tetrads (Te). In Onc. sphacelatum (A), the cells of middle layer and the tapetum (arrow) have become compressed, whereas in Oncsa. Gower Ramsey (B) the cells of middle layer and the tapetum have degenerated during meiosis. The pollen tetrads (Pt) of (C) Onc. sphacelatum and (D) Oncsa. Gower Ramsey contain a vegetative cell (arrow) and a generative cell (arrowhead) after pollen mitosis I. At anthesis, the pollen tetrads of Onc. sphacelatum (E) contain numerous lipid bodies (arrow) and protein bodies, whereas in Oncsa. Gower Ramsey (F), most mature pollen tetrads (Pt) have shrunk and degenerated. Scale bar = 10 μm. N = nucleus; Ed = endothecium.

Citation: HortScience horts 53, 9; 10.21273/HORTSCI13011-18

Sporad type observation.

Following the process of meiosis, each PMC normally produced a tetrad with four microspores in Onc. sphacelatum (Fig. 3A), whereas in Oncsa. Gower Ramsey, a considerable number of irregular sporad types were observed (Fig. 3B–D). In Onc. sphacelatum, 85.8% of normal tetrads were formed, whereas in Oncsa. Gower Ramsey, only 59.8% of normal tetrads were formed (Table 1). The frequency of abnormal sporads (e.g., dyad, triad, and sporads with micronuclei) in Oncsa. Gower Ramsey (40.2%) was higher than those in Onc. sphacelatum (14.2%).

Fig. 3.
Fig. 3.

The sporad type observation in Onc. sphacelatum and Oncsa. Gower Ramsey. The presence of nuclei is confirmed by the 4′,6-diamidino-2-phenylindole stain when viewed with a fluorescence microscope. The normal tetrad (A) of Onc. sphacelatum, whereas a dyad (B), an irregular tetrad (C) with micronuclei (arrows), and a triad (D) containing two large nuclei and one small nucleus (arrow) are commonly observed in Oncsa. Gower Ramsey. Scale bar = 10 μm.

Citation: HortScience horts 53, 9; 10.21273/HORTSCI13011-18

Table 1.

The number and percentage of sporad type observed in Onc. sphacelatum and Oncsa. Gower Ramsey.

Table 1.

Capsule setting by cross-pollination.

Capsule setting of 55% and 81.3%, respectively, was observed when Oncsa. Gower Ramsey was used as the female parent and Onc. sphacelatum and Gom. Java as the male parent. No capsule setting was observed when Oncsa. Gower Ramsey was used as the male parent and Onc. sphacelatum and Gom. Java as the female parent (Table 2).

Table 2.

Capsule setting of Oncsa. Gower Ramsey by cross-pollination.

Table 2.

Discussion

In this study, as compared with Onc. sphacelatum, the lower pollen viability and the higher abnormal sporads observed in Oncsa. Gower Ramsey (Tables 1 and 3) may be attributed to the irregularity of meiotic pairing. Oncsa. Gower Ramsey is an intergeneric hybrid that comprises three species, i.e., Onc. sphacelatum (2n = 56), Gom. flexuosa (Lodd.) M.W. Chase & N.H. Williams Sims (2n = 56), and Gom. varicosa (Lindl.) M.W. Chase & N.H. Williams (2n = 112) (Felix and Guerra, 2000), and the counts of chromosome number 2n = ca. 75–83 were observed in Oncsa. Gower Ramsey, suggesting a polyploid or aneuploid hybrid (Hu, 2001). The degeneration of sporads with unbalanced genomes has been reported in a number of primary hybrids in Phalaenopsis Blume (Arends, 1970) and Paphiopedilum Lindl. (Lee et al., 2011). Lee (1987) examined the meiotic behaviors and the sporad formation of different clones of an intergeneric orchid hybrid, Aranda Lucky Laycock (Arachnis hookerana Rchb. × Vanda tricolor Lindl.), and found that the fertile clones produced a higher frequency of dyads and tetrads without micronuclei, whereas the infertile clones produced more dyads and tetrads with micronuclei. Similarly, in Doritaenopsis orchids, the cultivars with high fertility were well correlated with the high frequency of normal tetrads (Bolanos-Villegas et al., 2008).

Table 3.

In vitro pollen germination of Onc. sphacelatum and Oncsa. Gower Ramsey.z

Table 3.

Like many orchids in the subfamily Epidendroideae, species within subtribe Oncidiinae have a compact pollinium that is composed of numerous tetrads (Pacini and Hesse, 2002). In our observations, at the time of anthesis, the pollinia within the anther locule of Oncsa. Gower Ramsey often appeared shriveled. To provide information about the pollinia abortion, histological studies were initiated to investigate the possible timing and tissues/cells involved in the abortion of pollinia during anther development. An interesting feature of this study revealed that the early degeneration of the middle layer and the tapetum may play an important role in the shriveled pollinia of Oncsa. Gower Ramsey. The tapetum is the innermost layer of the anther walls that contacts directly with the developing pollen and provides necessary nutrients and elements for pollen development and maturation (Pacini, 2010). In general, the tapetum begins to degenerate at the tetrad stage and is crushed completely by the bicellular pollen stage (Zhang et al., 2011) or the vacuolated microspore stage (Mizelle et al., 1989). Judging from our histological sections, the middle layer and the tapetum of Oncsa. Gower Ramsey became disorganized before meiosis (Fig. 1F). At the early tetrad stage, the middle layer and the tapetum of Oncsa. Gower Ramsey already disappeared (Fig. 2B). On the contrary, the middle layer and the tapetum of Onc. sphacelatum began to degenerate at the early tetrad stage (Fig. 2A) and fully disappeared at the bicellular pollen stage (Fig. 2C).

In our results of cross-pollination, 55% to 81.3% of capsule setting could be obtained when Oncsa. Gower Ramsey was used as the female parent, whereas no capsule setting could be observed when Oncsa. Gower Ramsey was used as the male parent (Table 2). It has been broadly reported that premature degeneration of tapetum usually causes male sterility. For example, in the somatic hybrids of Solanum commersonii Dun. and S. tuberosum Lindl., the early breakdown of tapetum and meiotic defects result in the male sterility (Conicella et al., 1997). In petunia, silencing of the tapetum-specific zinc finger gene may result in the precocious degeneration of tapetum and the subsequent pollen abortion (Kapoor et al., 2002). In the photoperiod-sensitive genic male sterility rice, the pollen abortion is associated with the premature degeneration of tapetum at the PMC stage (Shi et al., 2009). In snap bean, pollen sterility is caused by the premature degeneration of tapetum under the condition of high temperature (Suzuki et al., 2001). Tapetum is a short-lived cell layer of anther wall, and the progression of cell death in tapetum is an orderly and essential process for the normal pollen development, anther dehiscence, and pollen release and consequently guarantees the pollen fertility (Varnier et al., 2005; Wu and Cheung, 2000). The premature or delayed programmed cell death (PCD) of tapetum will result in the male sterility (Papini et al., 1999). In kiwifruit, male sterility was caused by a delayed PCD in the middle layer and tapetum (Giuseppina et al., 2013), whereas the premature PCD is detected in tapetum of the male-sterile lines of rice (Ku et al., 2003) and sunflower (Balk and Leaver, 2001). In Oncsa. Gower Ramsey, the middle layer and the tapetum lost their regular cell shape before meiosis, then followed by cell condensation, which is similar to the progression of premature PCD in tapetum observed in the male-sterile lines of rice (Ku et al., 2003). Further studies are needed to identify the possible role of PCD involved in the abnormal pollen development of Oncsa. Gower Ramsey.

In conclusion, we compared the ontogeny of the anther development in Oncsa. Gower Ramsey and the fertile diploid species, Onc. sphacelatum, and provide both the histological and cytological marks of the events causing the low fertility of Oncsa. Gower Ramsey. We propose that the abnormal meiosis caused by unbalanced genomes and the premature degeneration of the middle layer and the tapetum could probably result in the abnormal pollen development and the low fertility of Oncsa. Gower Ramsey.

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Contributor Notes

This work was supported by grants from National Museum of Natural Science, Taiwan, R.O.C., to Yung-I Lee, and the grant from Council of Agriculture, Executive Yuan, R.O.C., to Cheng-Jung Hu.

Corresponding author. E-mail: leeyungi@hotmail.com or leeyungi@mail.nmns.edu.tw.

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    Micrographs of the microspore mother cell (MMC) development of Onc. sphacelatum and Oncsa. Gower Ramsey. In (A) Onc. sphacelatum and (B) Oncsa. Gower Ramsey, their sporogenous cells (S) have dense cytoplasms with nuclei of great volume. Cell divisions in the primary parietal layer give rise to multilayered microsporangial walls that differentiate into the endothecium (Ed), the middle layer (M), and the tapetum (T) in (C) Onc. sphacelatum and (D) Oncsa. Gower Ramsey, and the sporogenous cells give rise to form MMCs. In Onc. sphacelatum (E), the chromatins of MMCs further condense in preparation for meiotic divisions, and the tapetal cells have elongated and possess prominent nuclei, whereas in Oncsa. Gower Ramsey (F), the cells of the middle layer and the tapetum are disorganized with the appearance of condensed nuclei. Scale bar = 10 μm.

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    Micrographs of the microgametogenesis of Onc. sphacelatum and Oncsa. Gower Ramsey. Meiotic divisions (arrowhead) result in the formation of microspore tetrads (Te). In Onc. sphacelatum (A), the cells of middle layer and the tapetum (arrow) have become compressed, whereas in Oncsa. Gower Ramsey (B) the cells of middle layer and the tapetum have degenerated during meiosis. The pollen tetrads (Pt) of (C) Onc. sphacelatum and (D) Oncsa. Gower Ramsey contain a vegetative cell (arrow) and a generative cell (arrowhead) after pollen mitosis I. At anthesis, the pollen tetrads of Onc. sphacelatum (E) contain numerous lipid bodies (arrow) and protein bodies, whereas in Oncsa. Gower Ramsey (F), most mature pollen tetrads (Pt) have shrunk and degenerated. Scale bar = 10 μm. N = nucleus; Ed = endothecium.

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    The sporad type observation in Onc. sphacelatum and Oncsa. Gower Ramsey. The presence of nuclei is confirmed by the 4′,6-diamidino-2-phenylindole stain when viewed with a fluorescence microscope. The normal tetrad (A) of Onc. sphacelatum, whereas a dyad (B), an irregular tetrad (C) with micronuclei (arrows), and a triad (D) containing two large nuclei and one small nucleus (arrow) are commonly observed in Oncsa. Gower Ramsey. Scale bar = 10 μm.

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