A Morphological and Histological Characterization of Male Flower in Chestnut ( Castanea ) Cultivar ‘ Yanshanzaofeng ’

Chinese chestnut (Castanea mollissima Blume.) is a widely distributed fruit tree and well known for its ecological and economic value. In order to evaluate obstacles to male reproductive in the C. mollissima, a morphological and histological characterization of male flower of chestnut cultivar ‘Yanshanzaofeng’ were examined by paraffin section technique and scanning electron microscopy. The results showed that male catkins with floral primordia were formed in the buds of one-year olds shoots in later April. Later, a protoderm, ground meristem and a procambium had differentiated in young anthers. Each young anther soon developed to four microsporangia. The anther wall layers developed completely by mid-May and consisted of one-cell-layered epidermis, one-cell-layered endothecium, two or three middle layers and one-cell-layered tapetum. The tapetum was of glandular type. Microspore mother cells underwent meiosis through simultaneous cytokinesis in later May and gave rise to tetrads of microspores, which were tetrahedrally arranged. Mature pollens contained two cells with three germ pores. Anthers were dehiscent and pollen grains shed by early June. Based our results, we did not find the abnormal male flower in the C. molissma cv ‘yanshanzaofeng’, indicating that male gametes were fertile and thus was considered as pollenizers.


INTRODUCTION
Chestnut (Castanea) belongs to the Fagaceae family which includes Quercus, Cyclobalanopsis, Castanopsis, Fagus, Lithocarpus and Trigonobalanus.Chestnut has been cultivated for food and timber since ancient times (Payne et al., 1983).In China, chestnut is one of the popular nuts because of positive health effects.It is a gluten-free flour source and contains essential dietary nutrients and minerals (Semih and Llknur, 2012).Chestnut production is particularly important source of income in rural regions, especially in Yan Mountain Region of Northern China (Qian et al., 2012).
Chestnut selection studies have been performed to determine superior genotypes in terms of yield and quality in Yan Mountain Region.Castanea mollissma cv 'yanshanzaofeng' was firstly selected from Yan Mountain Region and became an improved varieties in 1989 (Baotang, 2003).It was famous for ripening in the early September and developed quickly in Qianxi county of Yan Mountain Region.It cultivated area up to 42,000 ha (Yongmin, 2010).As we know, chestnuts are monoecious, bearing both staminate (male) and pistillate (female) flowers on the same tree (Botta et al., 1995).Male reproductive success was considered as the key factors responsible for fruit (Beverly and Mary, 1998;García-Mozo et al., 2001).Thus, an understanding of the male flower development is of basic importance to achieve high quality and consistently high production in chestnut orchards.In recently years, there have been some reports on the characteristics of the male flower development in Castanea (Botta et al., 1995;Ceriye and Arif, 2006;Yong-Qing et al., 2011).However, there was no concomitant description on morphological characterization of this cultivar 'Yanshanzaofeng'.Moreover, we have no information about the distribution of polysaccharides during the male gametophyte development in Castanea.In this study, we tried to verify the morphological and histological characterization of male flower in this cultivar, which may obtain basis information for appropriate management during flowering period and have the potential to improve nut yield and quality in the Yan Mountain Region.
This site is located in the warm temperate zone and semi-humid region, with a mean annual precipitation of 744.7 mm and a mean annual temperature of 10.9°C.The samples were collected about every week from April 2011 to June 2012.
Scanning Electron Microscopy (SEM).The materials were fixed in 2.5% glutaraldehyde, dehydrated through an ethyl-alcohol series (Zhuogong and Li, 2010) and then by freeze-drying treatment (Hitachi ES-2030, Japan).The materials were mounted on SEM stubs, coated with gold-palladium (Hitachi E-1010, Japan) and examined with a SEM (Hitachi S-3400N, Japan).

RESULTS
Morphological of male inflorescences: Initial development of staminate inflorescence primordial was observed in material collected in late April (Fig. 1A and  B).By early May, the apex of the structure which subsequently developed into a staminate inflorescence arose laterally on the shoot axis between the development leaf primordial and innermost bud scales.Within two weeks, primary, secondary and tertiary bracts were regularly present and six or so of the quaternary or perhaps higher order bracts, although these latter occur only in a median position on the upper and lower sides (Fig. 1C and D).By later May, then anthers were almost totally covered by tepals (Fig. 1E).In the early June, the staminate inflorescence has lengthened and appeared closer to its mature shape (Fig. 1F).At maturity, the tepals of the staminate flowers were reflexed, completely exposing the conspicuously lobed anthers (Fig. 1G).A few days prior to dehiscence, the mature anther was filled with pollen grains (Fig. 1G).The pollen grains were three germ pores (Fig. 1H).

Male flower development:
Formation of anther wall: The male flowers were tetrasporangiate anthers.At early stage of development, archesporial cells, which were recognizable by their large volume and conspicuous nuclei, differentiated below the epidermis of anthers (Fig. 2A).These cells divided periclinally to form outer primary parietal cells and inner primary sporogenous cells (Fig. 2A).The epidermal layer consisted of slightly elongated cell that developed into compressed epidermis cells (Fig. 2B).The primary parietal cells formed a subepidermal endothecium and two middle layers by periclinal and anticlinal divisions (Fig. 2B).
The outer secondary parietal cells by periclinal and anticlinal divisions.The endothecial cells elongated gradually, acquiring fibrous thickenings at the time of anthesis (Fig. 2K).The middle layers had a common histogenetic orign with the endothecium and it persisted until the tetrad stage and degenerated before forming two-celled pollen grains (Fig. 2J).The innermost layer gave rise to the tapetum, which partly originated from the ground tissue near the connective tissue and some of the tapetum cells had one to two prominent nuclei at the microspore mother cell phase (Fig. 2C).At the tetrad stage, tapetal cells elongated and lost close contact but still remained in their original position (Fig. 2H).They began to degenerate at uninucleate pollen stage and disintegrate at binucleate pollen stage.The endothecium comprised one layer.The middle layers appeared to undergo further divisions to form three layers.The anther wall prior to maturation was usually comprised of (5 to 6) cell layers, i.e., an epidermis, an endothecium, two or three middle layers and a layered tapetum.Thus, the tapetum was of the glandular type and wall formation conformed to the basic type as defined by Davis (1966).

Microsporogenesis and microgametogensis:
A row of sporogenous cells, produced by archesporial cells, gave rise to a mass of microspore mother cells by several mitotic divisions (Fig. 2B).Simultaneously with changes taking place in the wall of the microsporangia, the primary sporogenous cells underwent mitosis to form secondary sporogenous cells, form which microsporocytes were derived.Microsporocytes were identifiable by their large volume, dense cytoplasm and conspicuous nuclei (Fig. 2B).Meanwhile, a few polysaccharides appeared in the sporogenous cells (Fig. 2L).The microsporocyte underwent meiosis and the process of meiosis involved two cell divisions, such as meiosis I includes the leptonema (Fig. 2C), the diakinesis (Fig. 2D), the prophase (Fig. 2E) and metaphase (Fig. 2F).As a result, a microspore dyad was formed.A microspore tetrad was formed via the prophase II, for example metaphase II (Fig. 2G).At the tetrad stage many polysaccharides increased in the tapetal cell (Fig. 2M).Most of the microspores in the tetrads were tetrahedral (Fig. 2H).Callose deposition occurred at the onset of meiosis of pollen mother cells, reached a peak at metaphase II or anaphase II by enveloping the pollen mother cells or micro tetrads (Fig. 2H) and disappeared at end of meiosis.It was considered that the cycling of callose during microsporgensis played an important role in the protection of the pollen mother cells and released of microspores form the micro tetrads (Scott et al., 2004).
In contrary, simultaneous cytokinesis took place after pollen mother cell meiosis.
The microspore tetrads soon separated from each other and released form the microspores (Fig. 2I).A few polysaccharides existed at the microspore stage (Fig. 2N).Each microspore had a dense cytoplasm, conspicuous wall and a prominent and centrally placed nucleus (Fig. 2I).As the central vacuole developed, the nucleus took a peripheral position.At this stage, the microspore was uninucleate (Fig. 2J) which underwent a mitotic division to give rise to a binucleate cell.However, at the mature pollen, tapetal cells fully degenerated and many polysaccharides were accumulated in pollen wall (Fig. 2O).Mature pollen grains contained two cells with three germ pores (Fig. 1H).Anthers were dehiscent and pollen grains shed approximately on June 10.

DISCUSSION
The male development characters of C. mollissma cv 'yanshanzaofeng' are similar to Cyclobalanopsis (Hann-Chung and Tzu-Ming, 2005), Castanea (Cevriye and Arif, 2006) and Quercus species (Deng et al., 2008).The anther of this cultivar 'Yanshanzaofeng' is microdiodanges.The mature anther wall comprises an epidermis, a layer of endothecium, two middle layers and a single-layered tapetum.The tapetum during development of the anther wall is of the heteromorphic and glandular type.This type of tapetum was reported for some species in Quercus (Stairs, 1964;Robert, 1985;Deng et al., 2008).At about the time of pollen tetrads, the walls of the tapetal cells became indistinct and the tapetal cells degenerated at their original site.The tapetal cells degenerated completely at the stage of one-nucleate pollen grains and the nucleus near the wall.The cytokinesis of microspore mother cell meiosis is modified simultaneous type and tetrads are tetrahedral.The pollen grain is two-celled type with three germ pores.
The distribution of polysaccharides during the male gametophyte development of the C. mollissma cv 'yanshanzaofeng' displayed some distinct features.There was a close relationship between the change of polysaccharide with the supplement of nutrition during the development of pollen (Albertini et al., 1981).At first, a few polysaccharides appeared in the sporogenous cells.At the tetrad stage many polysaccharides increased in the tapetal cell.Subsequently, the tapetum began to degenerate.A few polysaccharides existed at the microspore stage.However, at the mature pollen, tapetal cells fully degenerated and many polysaccharides were accumulated in pollen wall.Thus, tapetal cells play the important role of providing nutrition substances for the development of microsporocytes (Scott et al., 2004).
Cytoplasmic male sterility should be taken into account as a factor influencing variations in chestnut production (Mckay, 1939;Jaynes, 1963;Omura and Akihama, 1980;Soylu, 1992;Sisco et al., 2001;Cevriye and Arif, 2006).Cytoplasmic male sterility are usually accompanied by anomalies tapetum (Kaul, 1988).In our anatomical investigation, we did not find the anomalies tapetum during microsporogenesis in the C. molissma cv 'yanshanzaofeng'.Therefore, it was indicated that male gametes were fertile and thus was considered as pollenizers.

CONCLUSION
This study provided basic information on different male reproduction aspects of C. mollissma and could shed light on the embryogenesis in this species.In C. mollissma cv 'yanshanzaofeng', the microgametogensis results in binucleate pollen as has been observed in other genera in Castanea.We did not find the abnormal male flower in the C. molissma cv 'yanshanzaofeng', indicating that male gametes were fertile and thus was considered as pollenizers.

ACKNOWLEDGMENT
The project was supported by the National Science and Technology Support (Grant No. 2013BAD14B0402), the Doctoral Foundation of Ministry Education (Grant No. 20120014110011) and the Public Welfare Project of Forestry Industry (Grant No. 201204401).

Fig. 1 :
Fig. 1: The morphological of male inflorescences in Castanea cultivar 'Yanshanzaofeng' A-B, Top view, late April, showing the staminate flower primordial.C, Early May, showing the anthers covered by primary, secondary and tertiary bracts.D, Middle May, showing the anthers covered by primary, secondary and tertiary bracts.E, Late May, showing the anthers covered by bracts.F, Top view, late May, is showing a mature staminate flower.G, Early June, a mature anther, showing pollen grains.H, Early June, mature pollen grains

Fig. 2 :
Fig. 2: Formation and development of microspore and male gametophyte in Castanea cultivar 'Yanshanzaofeng'A, The corners of the young anther development aarchesporial cell.B, Anther showing microspore mother cell with developed anther wall: epidermis layer, endothecium, two or three middle layers and inner tapetum.C, Anther showing central microspore mother cell at leptonema stage.D, Anther showing central microspore mother cell at diakinesis.E, Anther showing central microspore mother cell at meiosis I prophase.F, Anther showing central microspore mother cell at meiosis I metaphase.G, Anther showing central microspore mother cell at meiosis II metaphase.H, Anther showing microspore terrads arranged tetrahedrally and enclosed in callose.I, Anthers showing the microspores were released from tetrads (early microspore stage).J, Anthers at uninucleate microspore stage.K, Anthesis showing anther wall with stretched