megasporogenesis, microsporogenesis, and development of

HORTSCIENCE 54(10):1686–1693. 2019. https://doi.org/10.21273/HORTSCI14237-19

Megasporogenesis, Microsporogenesis,and Development of Female and MaleGametophytes of Ziziphus jujubaMill. ‘Zhongqiusucui’Fengxia Shao, Sen Wang, Juan Chen, and Rongyan HongKey Laboratory of Cultivation and Protection for Non-Wood Forest Trees,Ministry of Education, Central South University of Forestry and Technology,Changsha 410004, China; Key Lab of Non-wood Forest Products of StateForestry Administration, Central South University of Forestry andTechnology, Changsha, Hunan, 410004, China; and Engineering andTechnology Research Institute of Jujube Industry in Southern China,Central South University of Forestry and Technology, Changsha, Hunan,410004, China

Additional index words. Ziziphus jujuba Mill. Zhongqiusucui, mega-and microsporogenesis,male gametogenesis, female gametogenesis

Abstract. To investigate whether reproductive disorders exist in the sexual reproductionof Ziziphus jujuba Mill. ‘Zhongqiusucui’ and to understand the reproductive biology of‘Zhongqiusucui’ and genetic improvements in jujube trees, we used ‘Zhongqiusucui’flowers at different developmental stages as materials and conducted field andmicroscopic observations on the developmental pattern of mega- andmicrosporogenesis,as well as on the development of male and female gametophytes. The results show thefollowing. 1) From the inflorescence development stage to flowering, the grade 0 bud onthe inflorescence exhibited an increase in horizontal diameter, longitudinal diameter,peduncle length, and bud weight, but the rates of increase were different. From day 1 today 5 after the inflorescence had developed, floral buds mostly grew horizontally. Day 5was the floral bud flattening stage. From day 6 to day 8 after the inflorescence haddeveloped, floral buds mostly grew longitudinally, and day 8 was the floral bud enlargingstage. 2) The stamens of ‘Zhongqiusucui’ had five anthers, and there were four loculesper anther. The anther wall consisted of epidermis, endothecium, one- to two-layeredmiddle layer, and a secretory-type tapetum. In addition, the development of the antherwall belonged to the basic type. The cytokinesis of the microsporocytes was synchronous,the tetrads mostly arranged as a tetrahedron, and the mature pollen had three germpores, three grooves, and was bicellular pollen. During meiosis, the microsporocytes ineach locule were at the same phase and therefore exhibited synchrony. Among thedifferent anthers in the same floral bud, as well as the four locules in the same anther, themicrosporocytes had asynchronous meiosis. 3) The pistils in the ‘Zhongqiusucui’ had twoovaries, two anatropous ovules, inner and outer integument, crassinucellate tetradsformed by the meiosis of megasporocytes aligned linearly along the nucellus, megasporeat the chalazal end that developed into the functional megaspore, which underwentmitotic division three times and developed into the mature embryo sac containing sevencells and eight nuclei, and embryo sac development of the Polygonum type. 4) Theexternal morphology of the ‘Zhongqiusucui’ floral buds correlated with the internaldevelopmental stage of the male and female gametophyte. Therefore, the internaldevelopmental progress of the stamen and pistil can be determined by the externalmorphological characteristics of the floral buds.

Ziziphus jujuba Mill. ‘Zhongqiusucui’(Ziziphus jujuba Mill.) is a new cultivar bredin Hunan Province, and it has the character-istics of a large fruit size and high sweetness.‘Zhongqiusucui’ has a cylindrical-shapedfruit, thin skin, thick flesh, small pit, anaverage fruit weight of 13.8 g, and an ediblerate up to 97.1%. The fruit is juicy and haswhite-colored flesh, a fine and crispy texture,and light fragrance, and its soluble solidcontent is 31.76%, its total sugar content is29.41%, and its flavor is balanced in sweet-ness (Wang et al., 2009). Compared with

other major cultivars, such as Dongzao,Lizao, and Pingguodongzao in northernChina, as well as Jidanzao in the YangtzeRiver Valley, ‘Zhongqiusucui’ is mostlycultivated in southern China, which hasunique geographic and climate conditions.This fruit has many advantages, such as ahigh yield, high sugar content, rich flavor,good texture, strong adaptability, and easystorability and transportability, and it hasbecome the main cultivar in southern Chinafor fresh consumption. However, in fruitproduction, ‘Zhongqiusucui’ also faces dis-

advantages, such as severe abscission offlowers and fruits and easy fruit cracking.To improve fruit quality and fruit setting rate,it is necessary to carry out crossbreeding andgermplasm innovation to combine the advan-tages of other jujube cultivars. However,crossbreeding also introduces certain issues,such as seed abortion and nonsetting fruit.Research to identify the causes of seedabortions is urgently required because solv-ing this challenge is critical for breeding newcultivars and improving fruit production.

Any disruption of the pollen, embryosac, embryo, and endosperm developmen-tal process would result in seed abortion(Liang et al., 2005). This study investi-gated the processes of microsporogenesisand megasporogenesis and the develop-ment of male and female gametophytes.Our goal was to elucidate whether repro-ductive disorders exist in the sexual re-production of ‘Zhongqiusucui’ and toprovide a foundation for carrying outcrossbreeding.

Materials and Methods

Experimental siteThe experimental site was the jujube tree

experimental base at Central South Univer-sity of Forestry and Technology (QidongCounty, Hunan Province, China), which islocated at lat. 26.78�N, long. 112.12�E.

Experimental cultivarsFive-year old Z. jujuba Mill. ‘Zhongqiu-

sucui’ trees that had been transplanted oncewere used in this study. We selected treeswith good growth and developmental condi-tions, relatively uniform tree vigor, and nodisease or pests.

Experimental methodsSample observation and treatment. At

the inflorescence developing stage, a largenumber of inflorescences on the top ofdifferent bearing shoots at the middle ofthe canopy were selected and marked. Fiveinflorescences were selected and followedfrom the beginning of the inflorescencedeveloping stage. At 10 AM every day,pictures of grade 0 buds were taken usinga Canon macro camera (EOS 5D Mark IV,EF 100 mm f/2.8L IS USM; Canon, Tokyo,Japan) to record their growth and develop-ment. Twenty inflorescences were selected,and the grade 0 buds on the inflorescencewere measured every day using a Verniercaliper to record the horizontal diameter(the width of flower bud), longitudinaldimeter (the vertical height from the baseto the top of flower bud), and pedunclelength. From the marked inflorescences, 30flower buds were collected every day.Fifteen buds were immediately immersedin formalin–acetic acid–alcohol fixative(50% ethanol:acetic acid:formaldehyde =90:5:5). Samples were placed in a vacuumto pull air out of the tissue and then placedin a 4 �C refrigerator for storage. For theother 15 floral buds, the weights of single

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buds were measured using an electricalanalytical balance.

Microscopic observations. The routineparaffin section method (Li, 2009) was usedto generate sections of 8-mm thickness. Sec-tions were stained using the modified Ehr-lich’s hematoxylin staining method (Xuet al., 2008) and mounted with neutral gum.A DMi8 inverted microscope (Leica, Wet-zlar, Germany) was used to observe theprocesses of microsporogenesis and mega-sporogenesis as well as the development ofmale and female gametophytes. Photographswere taken to record the observations.

Results and Analysis

Observations of jujube morphologyduring the floral bud stage

The flowering period of single jujubeflowers began from the appearance of floralbuds and lasted until the enlargement ofovaries. Qu et al. (1989) divided the jujubeflowering process into three main stages: thefloral bud stage, flowering stage, and youngfruit stage. Three stages are included withinthe floral bud stage: floral bud appearance,inflorescence development, and floral budyellowing stage. The appearance of the floralbud stage occurred when the floral buds firstappeared at leaf axils. The inflorescence de-veloping stage occurred when the axillarybuds first appeared below one side of thegrade 0 buds on the inflorescence. The floralbud yellowing stage occurred when floralbuds turned yellowish green or yellow fromgreen. Based on previous observations andpreliminary experiments, we selected floralbuds that were at the inflorescence develop-ing stage, as well as those a few days past theinflorescence developing stage, as observa-tion objects for this study.

From day 1 at the inflorescence develop-ing stage, the marked grade 0 buds on theinflorescence were observed and photo-graphed (Fig. 1). On the day when the in-florescence developed (Fig. 1A), the grade 0buds were relatively small and half-covered

by bracts. Five sepals were not obvious. Onone side of the grade 0 buds, lateral younggrade 1 buds could be observed. As the grade0 buds gradually enlarged, their colors alsochanged. From day 1 to day 3, the grade 0buds were bright green-colored (Fig. 1A–C).On day 4, the color of the grade 0 budschanged to green (Fig. 1D), which slightlydarkened on day 5 (Fig. 1E). The green colorwas maintained from this point to day 9,when the grade 0 buds appeared to beyellowish green (Fig. 1I). On day 12, theyellow color started to become more obvious(Fig. 1L). On day 13, the floral buds wereenlarged to their maximum size and wereyellow-colored (Fig. 1M). A small fraction ofthe grade 0 buds began to open and blossom(Fig. 1N). From this point, all observed floralbuds blossomed.

To further study the growth and develop-mental pattern of the grade 0 buds on differ-ent days after the first appearance of theinflorescence, we measured and calculatedtheir horizontal diameter. The results areshown in Figs. 2 and 3.

As shown in Fig. 2, at different times atthe inflorescence developing stage, the grade0 buds exhibited an increase in horizontaldiameter, longitudinal diameter, pedunclelength, and bud weight. The horizontal andlongitudinal diameters of the floral buds didnot exhibit any significant difference on day 1at the inflorescence developing stage, whereasthey did show some differences in the follow-ing days. From day 1 to day 5 at the in-florescence developing stage, the budhorizontal diameter increased faster than thelongitudinal diameter, and this different rate ofincrease could also be confirmed by the ratioof these two values. As shown in Fig. 3, theratio of longitudinal diameter/horizontal di-ameter exhibited a decrease from day 1 to day5, indicating that during this period, the floralbuds were mostly elongated horizontally, andon day 5, the floral buds had a rather flattenedshape. In previous studies on jujube flowerdevelopment, this stage was called the flat budstage (Zeng, 1959). From day 5 to day 6, theratio of the increase in horizontal and longitu-dinal diameter stayed the same, followed by anincrease in the ratio until reaching its peak onday 8. This finding suggests that from day 6 today 8, the floral buds grew mostly in thelongitudinal direction. At this point, the floralbud longitudinal diameter was larger than itwas previously, and the shapes of the budswere rounder. This stage was referred to as thefloral bud enlarging stage (Zeng, 1959). Fromday 8 onward, the ratio of longitudinal andhorizontal diameters exhibited a small degreeof fluctuation. Figure 2 also shows that thelength of the peduncle had a rate of increasesimilar to those of the longitudinal and hori-zontal diameter from day 1 to day 6, whichwas followed by a rapid increase, and thepeduncle length eventually exceeded the budlongitudinal diameter on day 9. From day 7 today 13, the length of the peduncle continued agradual and steady increase but stayed shorterthan the horizontal diameter of the bud. Thefloral bud weight began to rapidly increase

from day 4, at a rate faster than all the otherparameters. This result may be because on day4 at the inflorescence developing stage, indi-vidual parts of the floral buds began to fullydifferentiate, and the accumulation of nutri-ents was faster.

Microsporogenesis and malegametophyte development

Anther wall development. The anther of‘Zhongqiusucui’ contained four locules. Dur-ing the early stages of development, thestamen primordia formed anther primordia(Fig. 4A), and anther differentiation had notyet begun. A layer of epidermis was on theoutside of the anther primordia. Underneaththe epidermis, a group of actively dividingcells was observed. Because cells in the fourcorners of the anther were dividing morerapidly, the anther developed into a four-angled shape. Under the epidermis of eachangle, archesporial cells were differentiated.Many archesporial cells with dense cyto-plasm were seen on the cross sections(Fig. 4B). After the formation of archesporialcells, they divided periclinally and furtherdifferentiated into primary parietal cells andprimary sporogenous cells (Fig. 4C and D).The primary parietal cells continued to dividepericlinally and vertically and produced aseries of cells arranged in concentric circlesof usually three to five layers. Together withthe outmost epidermis, they formed the an-ther wall (Fig. 4E and F). The anther wall of‘Zhongqiusucui’ fully differentiated at themicrospore mother cell stage (Fig. 4G), and itcontained four layers: the epidermis, endo-thecium, middle layer, and tapetum (from theoutermost to the innermost layers). The mid-dle layer was a single layer of cells. Duringthe meiosis of the microsporocyte, cells in themiddle layer already began to differentiateand showed decreased storage substances,and the cells flattened and gradually degen-erated and were absorbed (Fig. 4H–O). Themiddle layer was absent in the mature antherof ‘Zhongqiusucui’ (Fig. 4U). During thedevelopment of the anther wall, the tapetumwas rich in nutrients and provided sufficientmaterial for the development of microspores.Thus, the tapetum was the most conspicuousduring the entire anther developmental pro-cess. These findings are similar to the resultsof Chunyan He (2009). During meiosis Iprophase of the microspore mother cell, thetapetum began to thicken radially (Fig. 4H).When meiosis began, the tapetum started todegrade gradually. At the stage of microsporetetrads, the tapetum began to disintegrate insitu (Fig. 4P) and continued degrading duringthe mononucleus stage. This led to theseparation of the tapetum from the middlelayer of the anther wall (Fig. 4Q and R).When the pollen of ‘Zhongqiusucui’ wasmaturing, the tapetum started to dissolveand degenerate and eventually disappeared.As a result, the anther wall of ‘Zhongqiusu-cui’, originally with four layers, containedonly two layers at this point: the endotheciumand epidermis (Fig. 4U). The anther walldevelopment in ‘Zhongqiusucui’ belonged to

Received for publication 5 June 2019. Accepted forpublication 8 July 2019.The key research and development project ofHunan Province ‘‘Research and Demonstration ofTechnology for Improving Quality, IncreasingYield and High Value Utilization of Woody GrainTree Species in Hunan Province (2018NK2043),’’the Key Research and Development Project ofHunan Province ‘‘Quality Innovation and KeyTechnologies of Main Nonwood Forest Speciesin Hunan Province (2016NK2147),’’ and the Post-graduate Science and Technology Innovation FundProject of Central South University of ForestryScience and Technology ‘‘Research on EmbryoAbortion Mechanism of Fresh-eating Jujube inSouth China (CX2016A02)’’ provided financialsupport for the conduct of the research and prep-aration of the article. Thanks are due to Juan Chenand Rongyan Hong for assistance with the exper-iments, and to Professor Sen Wang for valuablediscussion.S.W. is the corresponding author. E-mail:[email protected].

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the basic type (Davis, 1966). Based on themorphological differences of tapetum duringits later stages of development, Davis (1966)divided tapetum into two types: the plasmo-dial type and secretory type. The degenera-tion and dissolution of the tapetum in‘Zhongqiusucui’ occurred at its original lo-cation. therefore, its nucleus and cytoplasmalso dissolved at the original location withoutchanging. Thus, the tapetum in ‘Zhongqiu-sucui’ was the secretory type, which isconsistent with that in ‘Dongzao’ (Huanget al., 2017) and ‘Jinsi No.4’ (Zou et al.,2013).

Microsporogenesis. The archesporial celldivided and formed the primary sporogenouscell, which then generated secondary spo-

rogenous cells through multiple rounds ofmitosis. The secondary sporogenous cell con-tinued to develop into microspore mothercells that had characteristics of a large cellvolume, large nucleus, and dense cytoplasm(Fig. 4G). The microsporocyte sequentiallywent through meiosis I prophase (Fig. 4H),metaphase (Fig. 4I), anaphase (Fig. 4J), andtelophase (Fig. 4K) and then completedmeiosis I and formed two daughter nuclei.Without forming a cell wall between the twodaughter nuclei, the cells entered meiosis IIand went through prophase (Fig. 4L), meta-phase (Fig. 4M), anaphase (Fig. 4N), andtelophase (Fig. 4O), and then tetrads wereformed (Fig. 4P). The tetrads were mainlytetrahedrally shaped, and a few were sym-metrical. Newly formed four haploid micro-spores were surrounded by a shared callosewall, and each microspore lacked its owncell wall. When tetrads subsequently beganto degenerate, the callose wall graduallydissolved. In tetrad microspores, the cellwall was formed and thickened, therebyleading to the development of free micro-spores (Fig. 4Q). The materials of the pollenwall were provided only by the cytoplasm ofmicrospores during the tetrad stage. after therelease of microspores, the wall materialscame from not only the microspores them-selves but also the tapetal cells (Hu, 2005).The meiosis process of microspores showedthat cytokinesis was simultaneous.

The microsporocytes in each locule wereat the same stage during meiosis andexhibited synchrony. In different anthers onthe same floral bud as well as the four loculeson the same anther, the meiosis of themicrospore mother cells was asynchronousand showed a difference of two to five phases.As shown in Fig. 5A–C, in the same floralbud, one anther was at the prophase ofmeiosis I (Fig. 5C), while another antherwas at the tetrad stage (Fig. 5B). In Fig. 5D,in the same anther, one pollen sac was at theanaphase of meiosis I while another one wasat the telophase of meiosis II.

Male gametophyte development. The de-velopment of male gametophytes in ‘Zhong-qiusucui’ began with microspores. Becausethe callose in the tetrad of microspores de-graded, dissolved, and disappeared, the mi-crospores were gradually released from thetetrad and formed haploid microspores. Theformation of haploid microspores markedthe beginning of microspores entering themale gametophyte developmental stage.When microspores were first released fromthe tetrad, they had thin cell walls, densecytoplasm, nucleus in the middle of the cell,and an overall small cell volume (Fig. 4Q).Microspores absorbed nutrients from theirsurrounding tissues and organs during theirdevelopment, which promoted the furtherdevelopment of microspores into haploidmicrospores with an increased cell volumeand enlarged nucleus (Fig. 4R). During thedevelopment of microspores, three germpores formed on the surface on the micro-spores, and they functioned as channels fornutrients provided by the tapetum to enter

Fig. 1. Morphological development characteristics of flower buds at the inflorescence developing stage.(A) The grade 0 bud on the inflorescence in day 1 (arrow). (B) The grade 0 bud on the inflorescence onday 2 (arrow). (C) The grade 0 bud on the inflorescence in day 3 (arrow). (D) The grade 0 bud on theinflorescence on day 4 (arrow). (E) The grade 0 bud on the inflorescence on day 5 (arrow). (F) Thegrade 0 bud on the inflorescence on day 6 (arrow). (G) The grade 0 bud on the inflorescence on day 7(arrow). (H) The grade 0 bud on the inflorescence on day 8 (arrow). (I) The grade 0 bud on theinflorescence on day 6 (arrow). (J) The grade 0 bud on the inflorescence on day 10 (arrow). (K) Thegrade 0 bud on the inflorescence on day 11 (arrow). (L) The grade 0 bud on the inflorescence on day 12(arrow). (M) The grade 0 bud on the inflorescence on day 13 (arrow). (N) The grade 0 opening floweron the inflorescence on day 13 (arrow).

Fig. 2. Changes of Zizyphus jujuba Mill. ‘Zhong-qiusucui’ bud size with time.


microspores. As microspores continued ab-sorbing water and nutrients from the tapetum,their cell volume increased, with a large

vacuole forming in the center of the cell. Thenuclei gradually moved to the cell periphery,and microspores entered the uninucleate mi-crospore stage (Fig. 4S). After this stage, thehaploid cell underwent an asymmetric mitoticdivision into diploid cells, with the larger cellas the vegetative cell and the smaller cell as thegenerative cell (Fig. 4T). As the pollen grainsincreased in volume, the cell wall between thegenerative cell and the vegetative cell disap-peared. The generative cell gradually migratedinto the cytoplasm of the vegetative cell andformed bicellular pollen, where the generativecell was round shaped with a relatively largenucleus. The bicellular pollen continued todevelop into a mature pollen grain that had atriangle shape, three germ pores, and threegrooves (Fig. 4U). The mature pollen grainis bicellular. During the development of mi-

crospores, we observed abnormal microsporedevelopment. Aborted microspores shrunk insize and contained no nucleus (Fig. 5E). Pollenwas absent in a few pollen sacs and presenteddegenerated and disappeared tapetum layers(Fig. 5F).We also observed an abnormal pollensac, as shown in Fig. 5G. One anther had threelocules at the uninucleate microspore stage,and one pollen sac presented no microsporesand was filled with an unidentified substance.In addition, the same situation as mentionedpreviously was observed in the pollen sac ofthe entire anther (Fig. 5H). These observationsof microspore development had not been pre-viously reported in other jujube cultivars. Wespeculated that this development pattern maybe due to abnormalities in the tapetum. how-ever, detailed causes have not been elucidatedand further studies are required.

Megasporogenesis and femalegametophyte development

Megasporogenesis. The timing of mega-sporogenesis in ‘Zhongqiusucui’ was obvi-ously later than that of microsporogenesis.We did not observe ovule primordia in theovary until microspores entered meiosis.The ovule primordia initiated from theplacenta of the ovary wall (Fig. 6A). Theapical cells in the ovule primordia furtherdifferentiated and formed the nucellus(Fig. 6B and C). Under the nucellus epider-mis, one cell differentiated into the arche-sporial cell (Fig. 6D). This archesporial cellwas different from other cells in the nucellusbecause of its large volume, dense cyto-plasm, and inclusion of an obvious nucleus.Next, the cells in the nucellus tissue differ-entiated into inner and outer integuments.The outer integument developed morequickly than the inner integument. there-fore, it gradually surrounded the nucellusand the inner integument. In later stages, oneside of the outer integument accelerated itsdevelopment, which caused the ovule togradually bend and eventually develop intoan anatropous ovule (Fig. 6E and F). Therewere two ovules in most of ovaries, oneovule in each chamber of the ovary. Mean-while, we observed three ovules in a fewnumbers of ovaries (Fig. 7A and B)

The archesporial cell in the nucellus di-vided periclinally and produced a parietal celland a sporogenous cell (Fig. 6E), with thelatter functioning as the megaspore mothercell. The megaspore mother cell underwentmeiosis I and formed dyads (Fig. 6E), whichthen continued to divide and formed fourmegaspores arranged linearly along the nu-cellus (Fig. 6F). Three megaspores close tothe micropyle gradually degenerated. Onlyone megaspore at the chalazal end becamethe functional megaspore. Generally, basedon the development of the archesporial cell,ovules can be divided into two forms: thetenuinucellar and crassinucellar forms. Thetenuinucellar ovule refers to the megasporemother cell that is directly underneath thenucellus epidermis without any parietalcells. In the crassinucellar ovule, the mega-spore mother cell was separated from the

Fig. 3. Variation of the ratio of longitudinal andtransverse diameters of jujube buds with time.

Fig. 4. Microsporogenesis and male gametophyte development. (A) Anther primordium. (B) Archesporialcell. (C) Periclinal division of archesporial cell. (D) Differentiation of anther inner wall andsporogenous cell. (E) Differentiation of anther inner wall and primary sporogenous cell. (F) Earlystage tapetum and secondary sporogenous cell. (G) Completion of anther wall development andmicrosporocytes. (H) Meiosis I prophase. (I) Meiosis I metaphase (blue arrow), early anaphase (blackarrow), and late anaphase (red arrow). (J) Meiosis I late anaphase (blue arrow). (K) Meiosis I telophase(blue arrow). (L) Meiosis II prophase (blue arrow). (M) Meiosis II metaphase (blue arrow). (N)Meiosis II anaphase (blue arrow). (O) Meiosis II telophase (blue arrow). (P) Tetrad phase (blue arrow).(Q) Microspores released from tetrads (blue arrow). (R) Early uninucleate microspore stage (bluearrow). (S) Late uninucleate microspore stage (blue arrow). (T) Binucleate stage (blue arrow). (U)Mature pollen grain, dense cytoplasm, with darker staining. (V) Mature pollen grain stage, crackedbetween two locules on the anther, pollen grain ready to disperse.


nucellus epidermis by one or a few layersof parietal cells (Hu, 2005). The megasporemother cell in ‘Zhongqiusucui’ was separatedfrom the nucellus epidermis by multiple layersof cells. therefore, its ovule belonged to thecrassinucellar form.

Female gametophyte development. Thefunctional megaspore further developed into

a uninucleate embryo sac with a large nucleusand an increased volume (Fig. 6H). Theuninucleate embryo sac underwent mitoticdivision once and formed two nuclei, whichwere pushed by the vacuole and migrated totwo poles to form the two-nucleate embryosac (Fig. 6I and J). Another mitotic divisionresulted in a four-nucleate embryo sac

(Fig. 6K and L). The four nuclei on bothsides of the embryo sac underwent anothermitotic division and formed the eight-nucleate embryo sac, with four nuclei atthe micropyle end and the other four nucleiat the chalazal end. One nucleus from themicropyle end migrated downward, onenucleus from the chalazal end migratedupward, and they together formed two par-allel nuclei at the center of the ovule. Thestage of the eight-nucleate embryo saclasted only briefly, and cell differentiationbegan quickly after. As the eight-nucleateembryo sac volume increased and cellulari-zation completed, the embryo sac entered itsmature stage (Fig. 6M–O). In the matureembryo sac, the egg cell and two synergidcells were at the micropyle end, and threeantipodal cells were at the chalazal end inthe nucellus tissue. Two polar nuclei were inthe center of the embryo sac. The matureembryo sac laid on top of the nucellus. Astalk-like structure that served as a pedestalformed underneath the embryo sac in thenucellus tissue of the ovule. From the de-velopmental pattern of the embryo sac in‘Zhongqiusucui’, we concluded that thestructure of its embryo sac was the Polygo-num type.

We observed some abnormally developedembryo sacs during female gametophyte de-velopment. Some embryo sacs had nocomplete eight nuclei, or scattered with approx-imately two to four irregular nuclei, whichwere often degenerated, with the absence ofsingle or multiple antipodal cells, egg cells,and helper cells (Fig. 7C–F). As shown inFig. 7C, there were only polar nucleus andegg cells in the embryo sac, and there wereno antipodal cells and helper cells in theadjacent sections of the embryo sac. Theembryo sac lacked antipodal cells, helpercells, egg cells, and another polar nucleus inFig. 7D. In Fig. 7E and F, the embryo sacwas scattered with approximately two tothree irregular nuclei, with the absence ofantipodal cells, helper cells, egg cells, andpolar nuclei. Antipodal cells were notobserved in most abnormally developedembryo sacs. Antipodal cells generally de-generate at the mature embryo sac stage orshortly after fertilization (Hu, 2005). There-fore, in this study, antipodal cells degener-ated earlier. There were only traces ofdegenerated cells (Fig. 7G and H), or only asmall number of inclusions, or completelyempty (Fig. 7I and J) in some other embryosacs. Because of the abnormal developmentof the embryo sac, the ovule loses its vitality.These embryo sacs were considered abnor-mal. As a result, although somewhat fullovules in the ovary locules were visible,under the microscope, these ovules wereempty sacs with only the integument.

Abnormal ovules also were observed.Some ovules were deformed early in devel-opment (Fig. 7K and L) or aborted (Fig. 7Mand N). There were also some other ovuleswith hypertrophic nucellus protruding out ofthe integument and containing abnormallydeveloped embryo sacs (Fig. 7O).

Fig. 5. Simultaneous phenomenon and abnormal microspore development. An = anther; AC = antherchamber. (A) Two anther in one bud. (B) Anther 1 of the bud in Fig. 1, staying tetrad phase. (C) Anther2 of the bud in Fig. 1, staying meiosis I prophase. (D) In the same anther, a pollen sac in meiosis I lateanaphase (blue arrow), and another at the meiosis II telophase (red arrow). (E) Abnormally developedpollen. (F) Empty pollen sac. (G) In the same anther, two2 chambers developed normally, and in earlyuninucleate microspore stage (black arrow). The other two did not develop normally. There were nomicrospores and filled with some unknown plasmids in one of the other two chambers (blue arrow).Microspores in the other one were abnormal development and surrounded by unknown plastid closely(red arrow). (H) Abnormal anther filled with unknown plastids.

Fig. 6. Megasporogenesis and female gametophyte development. (A) Ovule primordium. (B) Ovuleprimordium differentiation. (C) Ovule primordium differentiation. (D) Archesporial cell (arrow). (E)Peripheral cells and sporogenic cells (arrow). (F) Dyad period (arrow). (G) Tetrad period. The threemegaspores near the micropyle are degenerating (arrows). (H) Mononuclear embryo (the arrowindicates a functional megaspore). (I, J) Two-nucleate embryo sac (arrow). (I) The chalazal endnucleus (arrow). (J) The nucellar end nucleus on all other surfaces of the same embryo sac (arrow). (K,L) Period of tetranuclear embryo sac: two sections of the same embryo sac. (K) The binucleus near thechalaza and micropyle, respectively (arrow), and (L) the other binucleus near the chalaza andmicropyle, respectively (arrow). (M, L, O) Mature embryo sac period: Three aspects of the sameembryo sac, respectively. (M) Antipodal cells near the chalaza (arrow). (N) The oocyte near themicropyle (arrow), the central cell containing two polar nuclei (arrow), and antipodal cells (arrow), andone antipodal cell near the chalaza (arrow). (O) Two synergid cells near the micropyle (arrow).


Correlation between the size andmorphology of floral buds with stagesduring male and female gametophytedevelopment

As shown in Table 1, in ‘Zhongqiusucui’,the development of the stamen and pistil wasasynchronous. However, as a hermaphroditicplant, the developmental progress of bothmale and female gametophytes in the samefloral bud could be observed simultaneously.The stamens developed earlier than the pistilsthroughout the entire development process ofmale and female gametophytes. When mi-crospores began meiosis, the ovule primordiafirst started to appear in the pistil. When thehaploid microspores were at the center or atthe periphery, we first observed the mega-spore mother cell, which was followed by theaccelerated development of the embryo sac.When pollen matured, the embryo sac hadnot reached its maturity. Until 2 to 3 d afteranther burst, the embryo sac became mature.We combined and analyzed the observationsof floral bud morphology from the inflores-cence developing stage to flowering stagewith the development of male and femalegametophytes within floral buds. We foundthat the floral bud morphology of ‘Zhongqiu-sucui’ was correlated with the developmentof male and female gametophytes. In otherwords, we could speculate on the develop-mental condition of male and female game-tophytes from the developmental age of floral

buds, phenophase, bud size, and externalmorphology.

Conclusions and Discussion

From the inflorescence developing stageto flowering stage, the development of thefloral bud stage was normal. The grade 0floral buds in the inflorescence exhibited anincrease in horizontal and longitudinal di-ameter, peduncle length, and bud weight,although the rate of increase of these param-eters differed. From day 1 to day 5 of theinflorescence developing stage, the horizon-tal diameter of the floral buds increased at ahigher rate than the longitudinal diameter.Floral buds mainly elongated horizontally. Atday 5 from the development of inflorescence,the floral bud flattening stage was observed.From day 6 to day 8 at the inflorescencedeveloping stage, floral buds grew mainly inthe longitudinal direction. Day 8 marked thebeginning of the floral bud enlarging stage.This finding is different from the observa-tions by Qu et al. (1989), who stated that ittook �20 d from the inflorescence develop-ing stage to floral bud yellowing stage. Onepossible reason for this discrepancy could berelated to the different jujube cultivars. In thestudy by Qu et al. (1989), experimentalcultivars were Lingzao, Pozao, Mayazao,Huizao, Hupingzao No. 1 and No. 2, andSuanzao, which were different from the

cultivar used in this study, that is, the ‘Zhong-qiusucui’. Another possible reason could bethe different climates between southern andnorthern China. In southern China, thetemperature is higher, and the daylight islonger, which shortens the time needed forfloral bud development and flowering. Thefloral bud weight started to rapidly increasefrom day 4 at the inflorescence developingstage, with a rate of increase that was higherthan that of the other three parameters. Thisfinding may be because on day 4, pistilsbegan to develop and the differentiation ofeach part in the floral bud was becomingmore complete. In addition, the accumula-tion of nutrients had also become morerapid. After the inflorescence developingstage, the color of the floral bud changedas follows: bright green - green - yellowishgreen - yellow. It took �12–13 d for thefloral bud to develop from the inflorescencedeveloping stage to the flowering stage.

The embryological characteristics of‘Zhongqiusucui’ were consistent with thosereported in ‘Dongzao’ (Huang et al., 2017),‘Jisi No. 4’ (Zou et al., 2013), ‘Changhong-zao’ (Wang, 1983), ‘Ningxiayuanzao’ (TianandMa, 1987), ‘Huizao’ (Zhang et al., 2004),and ‘Suanzao’ (Niu et al., 2011).The antherof ‘Zhongqiusucui’ had four locules, and theanther wall consisted of the epidermis, endo-thecium, one to two layers of middle layer,and tapetum. In addition, basic-type antherdevelopment and secretory-type tapetum de-velopment were observed. During the devel-opment of the anther wall, cells in the middlelayer gradually degenerated or disappeared.The cytokinesis of microspores was simulta-neous. The tetrads mainly had a tetrahedralarrangement, and a few had a symmetricalarrangement. Mature pollen grains werebicellular and presented three germ poresand three grooves. In the ‘Zhongqiusucui’anther, the microsporocytes in each loculewere at a similar phase during meiosis andpresented synchronous characteristics. How-ever, there was desynchronization occasion-ally. Among the five anthers within the samefloral bud, meiosis of the microspore mothercells was asynchronous. This asynchrony wasalso true among four locules within the sameanther. The difference could be two to fivephases. A related study showed that beforemeiosis started, cells in the same layer of theanther wall, as well as adjacent cell layers,were connected by plasmodesmata (Hu,2005). The microspore mother cells withinone pollen sac were connected by plasmo-desmata, through which solutes and otherfactors could move from one microsporemother cell to another. This was consideredthe reason for the synchrony in meiosis(Heslop-Harrison, 1966; Whelan, 1974).When microspore mother cells underwentmeiosis, the callose gradually deposited onthe wall of the microspore mother cells whileplasmodesmata connections were broken.The secreted callose existed between theprimary cell wall and cytoplasm. Micro-spores formed by meiosis were surroundedby a shared callose wall, and they were also

Fig. 7. Three ovules in one ovary and abnormally developed embryo sacs. (A) Cross section of three ovulesin one ovary. (B) Longitudinal section of two ovules in one chamber of ovary. (C) Abnormal embryosac with only polar nucleus and egg cells (arrows). (D) Abnormal embryo sac with only polar nucleusand one antipodal cell (arrows). (E, F) Abnormal embryo sac with two to three scattered irregularnuclei (arrows), missing antipodal cells, helper cells, egg cells and polar nuclei. (G, H) Abnormalembryo sac containing only the vestigial traces of a group of degenerated cells (arrow). (I) Abnormalembryo sac with a small number of inclusions (arrow). (J) Abnormal embryo empty sac (arrow). (K,L)Deformed ovule. (M) Degenerated abortive ovule. (N) Two degenerated ovules in one ovary (arrows).(O) Hypertrophic nucellus containing an abnormally developed embryo sac (arrow).


separated from each other by callose. Thus,microspore mother cells, tetrads, or micro-spores were separated or isolated, which ledto a lack of synchrony after meiosis started.However, whether plasmodesmata were pres-ent among the pollen sacs and anthers was notclear, although the development was syn-chronous among different pollen sacs andanthers before meiosis. As a result, structuralconnections cannot sufficiently explain thereasons for this synchronous phenomenon.thus, further studies are required to investi-gate the causes. This asynchronous meiosisof microspore mother cells was also observedin other jujube cultivars, such as Dongzao(Huang et al., 2017) and ‘Jinsi No. 4’ jujube(Zou et al., 2013). Many studies have focusedon the asynchronous phenomenon during themeiosis of microspore mother cells in otherplant species (Meng et al., 2009). Duringmicrosporogenesis and male gametophytedevelopment, we did not observe a largeamount of aborted pollen, indicating thatthe pollen in ‘Zhongqiusucui’ was fertile,and aborted pollen could not explain embryoabortion in the jujube fruits. Abnormal de-velopment of the pollen, pollen sac, andempty pollen sac was occasionally observed,which might be related to the disappearanceand degeneration of tapetum (He and Wu,2013; Zhang et al., 2008, 2014). The specificreasons need to be further studied.

‘Zhongqiusucui’ has two-locular ovaries,two ovules in most of the ovaries (only a fewovaries contain three ovules), anatropousovules, double integuments, and crassinucel-lar ovules. The megaspore mother cell un-derwent meiosis twice and formed tetradsthat aligned linearly along the nucellus.Three megaspores near the micropyle endgradually degenerated, and one megaspore atthe chalazal end became the functional mega-spore. Seventy percent of the plants studiedhad a monosporic embryo sac, in which onlyone megaspore differentiated into the func-tional megaspore, whereas the other threemegaspores did not differentiate or divide. Insome species, a differently positioned mega-spore differentiates into the functional mega-spore, indicating the origin of the functionalmegaspore is regulated by a flexible devel-opmental pathway. In plants such as Arabi-dopsis (Webbm and Gunning, 1990), callosedeposition is closely correlated with theselection of a functional megaspore. Thecallose wall exists as a physical barrier,blocking the material and information com-munication between megaspores and theirsurrounding nucellus tissue, which results inthe megaspores at the micropyle end beingunable to develop into a functional mega-spore. In Puccinellia tenuiflora, the numberof normally developed megaspores is be-tween one and three, and the position of themegaspore that eventually develops into thefunctional megaspore is not fixed but isusually the first or the second one at thechalazal end, with a higher percentage beingthe latter one. This finding indicates thatsome cell degeneration derived from mega-sporocyte meiosis is genetically determined.T

able

1.Correspondingrelationship

betweenflower

budsize

andexternalmorphologyandgam

etophyte

developmentofZ.jujubaMill.Zhongqiusucui.

Stages

Horizontal

diam

(mm)

Longitudinal

diam

(mm)

Colorandstate

Developmentstageofstam

enDevelopmentstageofpistil

Day

10.86–1.26

0.68–1.03

Lightgreen

Anther

primordium

Undifferentiated

Day

21.23–1.56

0.79–1.16

Lightgreen

Archesporial

cell

Undifferentiated

Day

31.45–1.87

1.01–1.34

Lightgreen

Parietalcellandsporogenouscell

Undifferentiated

Day

41.61–2.31

1.18–1.54

Green

Differentiationofanther

inner

wall;primary

sporogenouscellsare

divided

into

secondarysporogenouscells

Undifferentiated

Day

51.94–2.65

1.21–1.65

Green;flat

budstage

Com

pletionofanther

walldevelopment;

microsporocytesform

ed;

meiosisIprophase

ofmicrosporocyte

Ovule

primordium

Day

62.31–3.01

1.44–1.83

Green

MeiosisImetaphase-

IItelophaseofmicrosporocyte;

tetrad

form

ation

Ovule

primordium

Day

72.37–3.23

1.59–1.98

Green

Tetradperiod;

microspores

released

from

tetrads

Archesporial

cell-sporogenouscell

Day

82.39–3.44

1.78–2.12

Green;floralbudenlargingstage

Slopenucleuscentered-slopenucleusbeside

Sporogenouscell

Day

92.64–3.67

1.89–2.28

Yellowgreen

Slopenucleusbesidestage-binucleate

stage

Megasporocyte;dyad

period

Day

10

2.77–3.76

2.02–2.36

Yellowgreen

Binucleate

stage

Tetradperiod;

functionalmegaspore

Day

11

2.89–3.82

2.08–2.44

Yellowgreen

Binucleate

stage-mature

pollen

grain

Mononuclearem

bryo-two-nucleate

embryosac

Day

12

3.08–3.91

2.13–2.52

Yellow;floralbudyellowingstage

Maturepollen

grain;anther

beginsto

crack

Two-nucleate

embryosac-four-nucleate

embryosac

Day

13

3.11–3.98

2.18–2.59

Yellow;somegrade0buds

began

toopen

Maturepollen

grain;anther

dehiscence

Four-nucleate

embryosac-eight-nucleate

maturedem

bryosac


Predictably, megasporogenesis is correlatedwith the callose (Yang and Shen, 2003). InLycium barbarum, callose begins to accumu-late on the sidewalls of the megasporesduring meiosis. At the dyad stage, exceptfor the cell wall at the chalazal end that didnot have callose accumulation, the cell wallat the other locations exhibits a thickenedcallose wall (Yang et al., 2017). Megasporo-genesis in angiosperms has a distinct feature:after the megasporocytes undergo meiosis,only one cell develops into the functionalmegaspore, and other cells undergo regulardegeneration.

The mature embryo sac was formedthrough mitotic division of the single func-tional megaspore. The developmental patternof the embryo sac was the Polygonum type.However, the developmental pattern of theembryo sac in jujube is still controversial. Inthis study, we found that the developmentalpattern of the embryo sac was the Polygonumtype, which is consistent with the observationby Srinivasachar (1940). However, Tian andMa (1987) considered the developmentalpattern to be the Allium type. The discrep-ancy may be attributable to the geneticcharacteristics in different jujube cultivars,although the specific causes still need to befurther investigated.

Dichogamy refers to the maturation ofstamens and pistils in one plant or one flowerat different times or the male and femalesexual functions at different time intervals(Zhao et al., 2011). ‘Zhongqiusucui’ hashermaphrodite flowers, and the developmentof its stamen and pistil were asynchronous.Therefore, ‘Zhongqiusucui’ is dichogamous,which is mainly represented by stamens de-veloping earlier than pistils. When micro-spore mother cells began meiosis, ovuleprimordia first appeared in the pistils. In laterstages, the development of pistils accelera-ted, and it was a relatively long period untilthe two-nucleate embryo sac was formed.When the anther cracked open and started torelease pollen, the embryo sac had justmatured. Although the development of pistilswas later than that of stamens, pollination andfertilization were unaffected because 12 to16 h are usually required for pollen togerminate after falling onto the stigma(Wang, 2008). In addition, the germinatedpollen requires time to reach the embryo sac.Thus, during this time, the embryo sac hasdeveloped to its maturity. The asynchronousdevelopment of male and female organs is amechanism of dichogamy to effectively pro-long the pollination period (Gabriela et al.,2008). Similar phenomena have been ob-served in other plant species, such as Juglansregia (Zhao et al., 2011) and Castaneamollissima.

In ‘Zhongqiusucui’, the floral bud exter-nal morphology was correlated with the de-velopment of male and female gametophytesinternally. We can understand the develop-ment of stamens and pistils by observing themorphological characteristics of floral budsand their sizes, which is helpful for the timely

and accurate management of the floweringperiod, promotes the normal development ofpollen and embryo sac, helps in the pre-diction of flowering time, and assists inartificial pollination. Meanwhile, our studyalso provided a strong scientific foundationfor tissue culture of jujube pollen and embryosac (Niu et al., 2011; Zhang et al., 2004),polyploid breeding (Xi et al., 2014), and in-depth molecular biology studies.

The results from this study showed thatalthough some abnormalities were presentduring microsporogenesis, and male game-tophyte development in ‘Zhongqiusucui’,the normal development of male gameto-phyte was unaffected overall. ‘Zhongqiusu-cui’ can produce normal mature pollen.There were some abnormal embryo sacsduring megasporogenesis and developmentof female gametophytes. Even if the abnor-mal embryo sac is temporarily fertilized,there will be obstacles in the subsequentdevelopment process. Seeds without endo-sperm or small embryo seeds, or emptyincomplete seeds will be formed. or seedsdegenerate and disappear during develop-ment. It is speculated that a large number ofembryo sac abortion is part of the cause ofseed abortion. However, the factors that leadto embryo abortion and a lack of settingfruits may occur during pollination, fertil-ization, and embryo development. Furtherstudies must be performed to discover theexact causes.

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