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Megasporogenesis and development of female gametophyte in Chinese chestnut (Castanea mollissima) cultivar Yanshanzaofeng'.

Byline: Feng Zou Su-juan Guo Peng Xie Huan Xiong Wen-jun Lv Guang-hui Li


Chinese Chestnut (Castanea mollissima Blume) is used as a non-wood forest tree and well-known for its edible value. Some chestnut cultivars are particularly prone to erratic fruit set showing very low or even empty cupule for unknown reasons. To investigate the causes of lack of fruit set in them the megasporgensis and development of female gametophytes of C. mollissima Yanshanzaofeng' were evaluated by microscopy. The results are showed that Yanshanzaofeng' was diclinous with polycarpous compound pistils axile placentas and commonly two anatropous ovules in each loculament. An archesporial cell in the nucellus tissue would directly develop into a megasporocyte that subsequently formed the megaspore tetrads aftermeiosis. The functional megaspore near the chalazal end developed into mature embryo sac which was of Polygonum type. After double fertilization the proembryo developed into a mature embryo going through the clavate globular heart torpedo and cotyledon stages. The embryo development conformed to the Onagrad type and the endosperm development was nuclear. Abnormal embryo sacs or abortive ovules were observed in the ovary. Our results suggested that the abortive ovules is a likelycause of low seed set in C. mollissima Yanshanzaofeng'. Copyright 2014 Friends Science Publishers

Keywords: Megasporogenesis; Female gametophyte; Abortive ovule; Chestnut


Chestnut has been cultivated as an important fruit crop since ancient times (Payne et al. 1983). It is widely distributed in the temperate zones of the Northern hemisphere (Ertan2007) including East Asia North American and Europe particularly in China (Serdar et al. 2011). Chinese chestnut (Castanea mollissma Blume) is an economically important cultivated species being low in fat and good eating quality with generally medium in size (Wang et al. 2012). Chinesechestnut has been introduced into many countries due to itsbest adaptability for good cold hardiness and adequate tolerance to chestnut blight (Bounous and Marinoni 2005). In addition Chinese chestnut plays an important role in keeping soil and greening barren hills (Martin et al. 2012).Understanding the reproductive biology of the species in order to make accurate predictions about patterns of chestnut production will be useful in both seed orchard management and increasing nut production in natural or planted stands (Feijo et al. 1999). The research on reproductive biology of the plant is considered as the keyfactors responsible for generating seeds (Guerra et al.2011). Thus the developmental process of megasporogenesis and female gametophytes plays a prominent role in contributing to population maintenance and regeneration of important species (Qu et al. 2010). In recent years considerable attention has been paid to theembryology of Castanea species (McKay 1940 1942; Zhang 1986; Xu 1988; Botta et al. 1995; Shi and StAlsser2005; Zheng et al. 2009). Although these sexual processes have been discussed in different Castanea species the reasons for low fruit rate of sexual reproduction in C. mollissima remain unknown or incomplete. Moreover there was no concomitant description on anatomiccharacterization of cultivar Yanshanzaofeng'. In generalmany of the cultivated varieties are sterile or produce only avery few viable seeds. One possible cause being the morphological abnormalities observed in flowers. Therefore detailed studies of this cultivar's embryogenesis development is essential for solving the problem.In this study the process of chestnut sexual production is described to provide a complete description of megasporgensis and development of female gametophytes. Our aim was not only to identify the main causes of low fruit set in open-pollinated flowers of this cultivar but also to provide basic information that will be helpful in the development of chestnut production protocols to enhance nut quality and yield.

Materials and Methods

Pistillate flowers of the chestnut cultivars C. mollissmaYanshanzaofeng' at successive developmental stages werecollected from a 12-year-old tree in Qianxi ChestnutOrchard (Hebei Province China) (4021'57N11812'17E) at approximately 163m above sea level. C.mollissma Yanshanzaofeng' was first selected in Qianxicounty and became an improved varieties in 1989. It wasfamous for ripening in the early September and developed so quickly that it covered about 45000 hectare cultivatedarea in Qianxi county (Zou et al. 2013). The materials werecollected every 3-5 days in June-August and beingrepresentative of the chestnut population. The pistillateflowers were fixed in FAA (70% alcohol: formalin: aceticacid = 90: 5: 5 v/v) for 24 h at room temperature(Chehregani and Sedaghat 2009) dehydrated through anethyl-alcohol series (Chehregani et al. 2011) embedded in paraffin with a 58-60oC melting point and sectioned at a thickness of 10 m by a microtome (Leika RM2265Germany). The sections were stained with Heidenhain's ironaium haematoxylin (Yuan et al. 2011; Guo et al. 2014).Observations and photographs of sections were carried out using an BX-51 and BX-61 microscope (Olympus Japan).


Development of Ovule

Pistil flower emergence and development lasted for 4 months - from May to August. The ovule primordiaumbegan development in the early June (Table 1). Approximately in mid of June the cells of the nucellar epidermal layer and the subdermal layer divided anticlinally. Some nucellar epidermal cells around the base of the nucellus resulted in the primordium of the inner integument by rapid mitosis (Fig. 1A). The inner integument was initiated from dermal cells at the base of the ovule primordium earlier than the outer one. The outer integument always grew more slowly than the inner. The integument enclosed the nucellus and formed the micropyle (Fig. 1A). The nucellus beneath the megaspore mother cells continuously elongated and developed into mature finger- like shape (Fig. 1B). In the nucellus the archesporial cell without periclinal division formed a primary parietal cell that directly developed into a megaspore mother cell on the later-June (Fig. 1C).Thus the ovules in C. mollissma were anatropous bitegmic and crassinucellate. In most other Fagaceae the structure of the ovules of C. mollissma was similar to species of Quercus.

Development of Megasporogensis and Megagametogenesis

On June 20 a typical megaspore mother cell was first observed which could be easily recognized by its large nucleus with dense cytoplasm (Fig. 1C) (Table 1). After meiosis I and II a megaspore tetrad was formed. Usually near the chalazal megaspore developed into the functional megaspore while the other three megaspores were eventually degenerated (Fig. 1D). The first division of the

Table 1: Phenology of embryogensis development in C. mollissima

Developmental stage###Approximate time when###Time before (-) or

###stage began###after (+) pollination

Ovule primordiaum###Early June###-2 week

Inner and outer###Middle June###-1 week


Pollination###Middle June

Megaspore mother cell###Later June###+1 week

One-nucleate embryo sac###Later June###+1 week

Eight-nucleate embryo###Early July###+2 week


Fertilization###Early July###+2 week

Free nuclear endosperm###Early July###+3 week

Zygote###Middle July###+3 week

Globose embryo###Middle July###+4 week

Torpedo-shaped embryo###Later July###+5 week

Cotyledon-shaped###Later July###+6 week


Mature embryo###Early August###+7 week

functional megaspore the nucleus resulted in the formation of the two-nucleate stage (Fig. 1E). After these two nuclei underwent a second meiotic division giving rise to form a four-nucleate female gametophyte (Fig. 1F). An additional mitosis of these nuclei an eight-nucleate megagametophyte was formed in the early-July (Fig. 1G-I). An egg cell and synergid cell that consisted of the egg apparatus were located at the micropylar pole but the synergid cell were degenerated very soon (Fig. 1H). Two polar nuclei met in the middle of the embryo sac and fused to form a single large central cell before fertilization (Fig. 1G). Three antipodal cells were located at the chalazal pole and generally degenerated soon (Fig. 1I). Thus this development of embryo was sac 8-nucleate Polugonum type.

Development of Endosperm

In the July 6 samples after double fertilization the zygote remained undivided which was located at the micropylar pole (Table 1). Although we did not see fertilization of the egg cell presuming fertilization occurred ~3 d earlier based on the formation of large primary endosperm nucleus (Fig.2A). In the sample collected on July 9 repeated divisions of the primary nuclei developed into the free nuclear endosperm (Table 1). The free nuclei were peripheral to the central area of the embryo sac by dense cytoplasm connections (Fig. 2C). Therefore the endosperm conformed to the nuclear type.The zygote was at dormancy stage during and after the development of the free nucleate endosperm whereas the inner integuments progressively degenerated (Fig. 2C). The endosperm eventually became cellular on the July 18 (Fig.2D).

Development of Embryo

In the early-July the division of zygote was delayed until the free nuclear endosperm was formed (Fig. 2A-C). After a well-developed endosperm the zygote resumed rapid growth by meiotic division later in July. Repeated divisions of all cells resulted in a clavate pro embryo (Fig. 2B). During further development the embryo became young globose embryo (Fig. 2D) torpedo- shaped (Fig. 2E) and finally cotyledon-shaped (Fig. 2F). The mature embryo was formed around the earlyAugust (Table 1). It consisted of radicle hypocotyl piumule and cotyledon surrounded by a mass of partially digested endosperm (Fig. 2G). Therefore this embryogeny conformed to the Onagrad type.Abortion of the Other Ovules during Development

A total of 16~18 ovules were found from the ovary axis but in the same ovary ovules were at several stages of development. Generally at least one embryo sac successfully developed in the ovary but only one always matured into a seed. In the developmental process of the ovules that did not have visible embryo sacs were either undeveloped or aborted. Due to insufficient pollination and fertilization abortion of ovules happened both before and after the development of the female gametophyte. It was hard to discriminate between the normal and aborted ovules unless the larger embryo sac appeared in the ovules (Fig.2H). When the endosperm nucleus was present in the center of the enlarging embryo sac around 18 July (Fig. 2H) suggesting that the other ovules showed signs of abortion. The aborted ovules were due to degenerated embryo sac with darkly stained or empty embryo sac (Fig. 2H). Thus it was reasonable to assume that these ovules were going to abort.


The method of embryogenesis in C. mollissima was the same as those reported for other species of in terms of the anatropous bitegmic and crassinucellate ovules and the Polygonum type embryo sac (Xu et al. 1988; Botta et al.1995; Zheng et al. 2009). Embryo development in Fagaceae follows the Onagrad type as previously described in Castanen (Botta et al. 1995; Shi and StOsser 2005).It is generally known that the normal ovuledevelopment is important for high fruit set in orchards (Jia et al. 2008). It was reported that in C. mollissma each ovule might be fertilized and be capable of producing a seed (McKay 1942). It was proposed that all of the ovules that develop a normal embryo sac are potential seeds (Mogensen 1975). In our anatomical experiment although most of the female gametophytes developed normally some ovules were abortive. The absence of embryo sacs and the occurrence of empty embryo sacs accounted for abortion in other ovules. The observed anomalies in C. mollissima also were consistent with previous observations (Yao et al.1990; Shi and StOsser 2005). To our knowledge the ovule is the source of the megagametophyte and the progenitor of the seed (Reiser and Fischer 1993). Poor fruit set has been attributed to an undesirable environmental conditions or male sterility or female sterility (Julian et al. 2010; Huang et al. 2011; Guerra et al. 2011). However in our study wefound Yanshanzaofeng' not only grew under a goodhydrothermal condition in orchards but also exhibited malefertility (Zou et al. 2013). The low fruit set inYanshanzaofeng' could be partially explained by abortiveovules in the female gametophytes development (Shi andStAlsser 2005) and a high percentage of abortive ovules was identified as the major factor causing female sterility and possibly influenced the nut production in Yanshanzaofeng'orchards. But further studies should be performed to delineate whether the abortive ovules were caused by failure fertilization or programmed cell death in the future.In conclusion this study provided basic information on the sexual reproduction aspects of C. mollissmaYanshanzaofeng'. In C. mollissma themacrogametogenesis belongs to the Polygonum type theendosperm conforms to the nuclear type and embryo development follows the Onagrad type as has been observed in other genera in Castanea. Abnormal embryo sacs or abortive ovules were observed in the ovary suggesting that the same cellular mechanisms might act in somatic as well as in embryo rescue and embryogenesis induction. A large number of abortive ovules may influence yield in C. mollissma and further studies are needed to corroborate these results.


We would like to express our gratitude and appreciation to all those who gave us the possibility to complete this work. We thank Professor Xiang-yang Kang of Beijing Forestry University and Vice Professor Ling Zhang of Connecticut University. The project was supported by the grant (No.20120014110011; No. 201204401; No.2013BAD14B0402).


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Author:Zou, Feng; Guo, Su-juan; Xie, Peng; Xiong, Huan; Lv, Wen-jun; Li, Guang-hui
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9CHIN
Date:Oct 31, 2014
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