Floral Biology of Chinese Jujube ( Ziziphus jujube Mill . ) I : The Formation of Microspores and Development of Male Gametes

Ziziphus jujube Mill is an economically important fruit tree, cultivated on marginal lands on a commercial scale, especially in China. In order to elucidate the microsporogenesis and male gametogenesis formation in Chinese jujube, a cultivar named ‘Lizao’ was employed for microscopy analysis. The results are showed that ‘Lizao’ was four ventricles in each anther and the anther wall was 4 to 5 layers. Its primary anther wall consisted of the epidermis, 1-2 layers endothecium, middle and glandular tapetum layers. The development of the anther wall conformed to the basic type. The meiosis in the microspore mother cells were belonged to a simultaneous type and most of the microspores were arranged in a tetrahedron shape in the tetrads. In the later May, the shape of pollen was a triangular germination vale with three germinal furrow and the mature pollen was of 2-cell type. Floral emergence and development lasted for 1 months-from later April to later May. Based our results, we did not find the abnormal male flower in the Z. jujube ‘Lizao’, suggesting that microsporogensis development may not be factors in the low seed production in the Z. jujube. This study provides the basis for understanding the biological mechanism regulating sexual reproduction, thus expanding the prospects for Z. jujube breeding programs and for further molecular and genetic studies of this species.


INTRODUCTION
Chinese jujube (Ziziphus jujube Mill.), a member of the Rhamnaceae family, originated from China and has cultivated for thousands of years.It is an economically important fruit crop species in China, well-know for its fruits with high dietary value and medical value (Jinghua et al., 2012).Jujube fruit contains flavonoids, vitamins, amino acids, organic acids, polysaccharides and microelements and found useful in spleen diseases and nourishment of blood in Chinese system of medicine (Sunil, 2013).Z. jujube is also tolerant to extreme environmental conditions, including drought, high temperatures and saline water (Tel-Zur and Schneider, 2009).
As our knowledge, environment, sexual reproduction and nutrition influence the flower and fruit fallen.Sexual reproduction includes sporogenesis and gametophyte development, pollination and fertilization, embryo and endosperm development and each part will affect the fruit set percentage (Lillecrapp et al., 1999).The flower and fruit fallen of jujube is caused by low temperature or cloudy and drizzly days during blossom period which affect the development of sporogenesis and gametophyte, pollination and fertilization.The absence of nutrition also maybe the reason analyzed by cultivation theory (Hu et al., 1999;Guangfang, 2014).Therefore, it is significant to study the reproductive biology of jujube.However, only a few investigation of the sexual reproduction of this species have been reported (Srinivasachar, 1940;Wang, 1983;Tian and Ma, 1987;Zhenhuai, 2004;Yuan et al., 2009;Yufei et al., 2011;Zhihui et al., 2011).The lack of basic knowledge in sexual reproduction becomes even more evident in Chinese jujube trees.They are difficult, when not impossible, to grow in controlled conditions and seasonal flowering is not easy to overcome.This creates a strong experimental constraint and the necessity to develop most of the experimental work in just a few months.It's generally accepted that a better knowledge of the mechanisms of pollination and fertilization may result in better productivity (Feng et al., 2013).Thus, knowledge of embryology on the development of reproductive organs in Z. jujube is essential for solving these problems.
Embryological studies are often useful in encompassing investigation of virtually all the events relevant to sexual reproduction.Microsporogenesis and male gametogenesis in Z. jujube 'Lizao' has not previously been investigated.This present study aims to investigate microsporogenesis and male gametogenesis of Z. jujube 'Lizao' and provide basic information for our research projects.

MATERIALS AND METHODS
The experiment was conducted during the year 2010 and 2012 on eight years old Z. jujube 'Lizao' which was planted at 2×3 m distance.The mixed planting species are Z.jujube.'Guangyangchangzao', 'Jidanzao' and 'Jinsi NO.4'.The experimental site was the non timber forest garden in Zhuzhou campus of Central South University of Forestry and Technology, which longitude is eastern 113°09′50″ and latitude is northern 27°55′30″ and altitude is 70.00 m and the site is gentle slope with re soil (Yuan et al., 2009).
We photographed the Z. jujube 'Lizao' from bud to bloom by the Nikon P600 camera (Japan) (Fig. 1A to H).The samples were collected about every 7 days from March to June and being representative of the jujube population.The materials were fixed in formalin-acetic acid-alcohol (FAA; 5:5:90, v/v), dehydrated through an ethyl-alcohol series, embedded in paraffin with a 58-60°C melting point and sectioned at a thickness of 10 µm by the microtome (Zou et al., 2013).The sections were stained with Heidenhain's iron aium haematoxylin (Zou et al., 2014).Observation and photograph of sections were carried out using an BX-51 microscope (Olympus, Japan).Some sections were stained with 0.5% decolorized aniline blue in 0.1 M K3po4 for 3~12 h and observed under a fluorescence Bx-51 microscope to investigate callose deposition during microsporogenesis (Feng et al., 2013).

RESULTS
Formation of anther wall: Floral emergence and development lasted for 1 month-from later April to later May (Fig. 1 and Table 1).The archesporial stage of microsporogenesis was first observed in buds collected in later April (Fig. 1A).The archesporial cells appeared at the corners of the young anther (Fig. 2A).The anthers were tetrasporangiate in the early May (Fig. 2B).At an early stage of development, archesporial cells (which were recognizable by their large volume with conspicuous nuclei) differentiated below the epidermis of anthers.These cells divided periclinally to Pollen grains released form outer primary parietal cells and inner primary sporogenous cells (Fig. 2B).The epidermal layer consisted of slightly elongated cells that gradually developed into compressed epidermis cells (Fig. 2B).
The archesporical cells divided by periclinal divisions to form and outer layer of primary parietal cells and inner primary sporogenus cells.The primary parietal cells divided repeatedly by periclinal and anticlinal divisions to form a subepidermal endothecium and two middle layers (Fig. 2B).Cells of the outer layer again divided periclinally and anticlinally to form the massive nucellus.The inner cells form the sporogenous cells (Fig. 2B).The endothecial cells elongated gradually, acquiring fibrous thickenings at the time of anthesis (Fig. 3G).The middle layers had a common histogenetic origin with the endothecium and it persisted until the tetrad stage (Fig. 2F) and degenerated before forming two-celled pollen grains (Fig. 3F).The middle layers appeared to undergo further divisions to form three layers.The rich starch in cells of middle layers was digested and gradually it disappeared in the process, providing nutrition for the pollen development.The walls of endothecium became thickened after microspore formation (Fig. 2H) and when pollen matured, the endothecium showed one layer (Fig. 3H).The anther wall, prior to maturity, was usually comprised of five or five cell layers, i.e., a single epidermis, an endothecium, two middle layers and the tapetum.Thus, the wall formation was of the basic type (Davis, 1966).

Tapetum characteristics:
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 or two prominent nuclei at the microspore mother cell meiosis stage (Fig. 2C to E).The tapetal cells were uninucleate or binucleate and posed abundant cytoplasm (Fig. 3B).The tapetum reached maximal size during meiosis of mircrospores.At the tetrad stage, tapetal cells elongated and lost close contact but still remained in their original position (Fig. 2F to G).However, they began to degenerate at the stage of uninucleate microspore (Fig. 3C) and has totally disintegrated at the mature pollen grain stage (Fig. 3H).Therefore, the tapetum conformed to the glandular type as named by Davis (1966).

Microsporogenesis and microgametogensis:
The anther primordium began to form as a teat, composing of meristematic tissue enveloped by an epidermal layer.
Simultaneously with the changes taking place in the wall of the microsporangia, four groups of hypodermal cells differentiated near each of the four corners of the young anther (Fig. 2A).From the sections, a row of sporogenous cells deriving from 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, forming secondary sporogenous cells, from which microsporocytes were derived.Microsporocytes were discernable by their large volume, dense cytoplasm and conspicuous nuclei (Fig. 2B).The microsporocyte underwent meiosis which involved two cell divisions.
Meiosis I includes the prophase, metaphase, anaphase and telophase (Fig. 2C).As a result, a dyad of microspores was formed.A microspore tetrad was formed during prophase II, metaphase II, anaphase II and telophase II (Fig. 2D to E). Meiosis II in each dyad resulted in a microspore tetrad.A majority of the tetrads were tetrahedral (Fig. 2F).There was a thick callose wall surrounding a microspore tetrad (Fig. 2G).We found that 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 microtetrads (Fig. 2G) and disappeared at end of of meiosis.The callose cycling (or cellulose synthesis) during microsporgensis might play important roles in protecting the pollen mother cells from various environmental stresses and release of microspores from the microtetrads.In all sections, we observed that only the second divisions occurred with the formation of wall between microspores, thus the microsporgenesis was simultaneous.The microspores were soon separated from each other and released form the tetrads as uninucleate free microspores (Fig. 2H).Each microspore released from the tetrads had a dense cytoplasm, conspicuous wall, with a prominent and centrally placed nucleus (Fig. 3A).During further development, uninucleate nicrospores gradually increased their volumes and had more vacuoles (Fig. 3B to D).As the central vacuole developed, the nucleus took a peripheral position (Fig. 3E to F).The microspore development of could be divided into three periods: early-or mid-uninucleate (Fig. 3C to D), late-uninucleate (Fig. 3E to F) and binucleate.At this stage, the uninucleate microspore underwent an asymmetric mitotic division (the microspore mitosis) to give rise to two cells with distinct fates-the vegetative cell and the generative cell.The nucleus of the vegetative cell was round and large and generally confined at the center of the pollen grain, whereas the smaller generative cell was crescent-shaped and located close to the pollen wall.In the later May, mature pollen grains contained two cells with three germ pores (Fig. 3G to H). Anthers were dehiscent (Fig. 1G to H) and pollen grains were shed approximately on May 29 th (Table 1).

DISCUSSION
The embryological feature of Z. jujube are summarized as follows.The anther of Z. jujube 'Lizao' is microdiodanges.The mature anther wall layers comprises an epidermis, one layered endothecium, two middle layers and a single-layered tapetum.The cytokinesis during meiosis of its microspore mother cell is modified simultaneous type and tetrads are tetrahedral.The formation of the anther wall conforms to the basic type.The pollen grains are monocolpate and two-celled with three germ pores at shedding.Some striking features are found in Ziziphus: • The anther wall was usually comprised of five or five cell layers, i.e., a single epidermis, an endothecium, two middle layers and the tapetum.Davis (1966) (Xue et al., 2005).From the sections, we observed that the tetrads of Ziziphus is tetrahedral.• The tapetum during the anther wall development is of the heteromorphic and glandular type.This type of tapetum was reported in some species of Ziziphus (Tel-Zur and Schneider, 2009).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 when anthers dehisced.Tapetum's major functions include producing pollen wall components, nutrients for pollen development and enzymes for microspore release from tetrads.All nutrients reaching the sporogenic cells must pass through tapetal cells (Yan et al., 2010).In addition, callose deposition occurred at the onset of meiosis of pollen mother cells and disappeared at end of meiosis.The callose cycling (or cellulose synthesis) during microsporgensis might play important roles in protecting the pollen mother cells from various environmental stresses and release of microspores from the microtetrads (Li and Ma, 2006).• Microsporogenesis of Ziziphus is successive.As we known, male sterility might be an important factor influencing fruit production (Feng et al., 2013).Male sterility is the result of failure of formation or development of functional stamens, microspores or gametes.According to Yan et al. (2010), there were some instances of failure to develop normal pollen, probably caused by the abnormal development at three levels: the sporogenous tissue, tapetum layer and microspore.Male sterility are usually accompanied by abnormal callose deposition or anomalies in tapetum (Kaul, 1988).However, in our anatomical observation, we did not find the abnormal callose deposition and anomalies in tapetum during microsporogenesis in the Z. jujube 'Lizao', indicating that male gametes were fertile and male sterility was not the major cause of the low seed set in the Z. jujube cultivar 'Lizao'.Because of a lack of sufficient data from each family of Rhamnaceae, we are need to clarify embryological attributes for each family in the future.

CONCLUSION
In the present study we clarify the stages of the reproductive cycle and consider similarities with other Z. jujube plants.In Z. jujube 'Lizao', the microgametogensis results in binucleate pollen as has been observed in other genera in Ziziphus.Cytological and embryological characteristics of Z. jujube 'Lizao' were studied for the first time.The reproductive biology of the Z. jujube species is still poorly understood and various aspects remain to be studied in depth.Only when we know more about the reproductive biology of the Rhamnaceae will it also become possible to understand and control fruit yield and quality in Chinese jujube.

Fig. 2 :
Fig. 2: Formation of microspores and development of male gametophyte in Z. jujube 'Lizao'; (A): The archesporial cells appeared at the corners of the young anther (filament, petal); (B): Detail of an microspore mother cell wall showing the microspore mother cell coat and thin layer of callose (epidermis layer, endothecium, two middle layers and inner tapetum); (C): Detail of a late microspore mother cell wall at meiosis Ⅰtelophase.G, detail of a late microspore mother cell wall at meiosis II metaphase; (D): Detail of a late microspore mother cell wall at meiosis II telophase; (E): High magnification of D showing microspore mother cell w+ all at meiosis II telophase; (F): Details of microspores showing microspore terrads were tetrahedral and enclosed in callose; (G): Details of anther showing microspore terrads were enclosed in the callose layer under the fluorescence microscope; (H): Details of anther showing at free microspores cell stage Ac: Archesporial cell; An: Anther; En: Endothecium; Ep: Epidermis; Fi: Filament; Mi: Middle layers; Mmc: Microspore mother cell; Ms: Microspore; Mst: Microspore tetrad; Pe: Petal; Se: Septum; T: Tapetum

Fig. 3 :
Fig. 3: Formation of microspores and development of male gametophyte in Z. jujube 'Lizao'; (A): Details of anther showing at microspores cell stage; (B): Anthers at uninucleate microspore stage with stretched epidermis, fibrous thickened endothecium, relic of middle layers; (C): Details of anther showing at stage of central nucleus microspore; (D): High magnification of C showing at stage of central nucleus microspore; (E): Details of anther showing at stage of late-nucleus microspore; (F): High magnification of C showing at stage of late-nucleus microspore; (G): Details of anthesis showing anther wall was composed of epidermis, endothecium and mature pollen grains; (H): High magnification of G showing epidermis, endothecium and mature pollen grains En: Endothecium; Ep: Epidermis; Ms: Microspore; Pg: Pollen grains; Se: Septum; T: Tapetum