Cotyledonary Embryos Research Articles

Flower morphologic anatomy and embryological characteristics in Chrysanthemum multicaule (Asteraceae)

Abstract Chrysanthemum multicaule is an annual herbaceous ornamental species. The inflorescence is gynomonoecious and consists of bisexual tubular florets and female ray florets. The pistils consist of two stigmas which are of the open type with a hollow stylar canal. At the base of the tubular floret style, the pistil is surrounded by oil gland cells. The anthers are tetrasporangiate and the young anther wall is composed of epidermis, endothecium, middle layer and tapetum. The mature anther wall comprises only thickened endothecium after the release of the pollens. In the tubular florets, simultaneous microsporocyte meiotic cytokinesis results in mostly tetrahedral with a small proportion of decussate tetrads. The mature pollen grain is tricellular. The ovules are unitegmic and tenuinucellate, and the nucellus degenerates during the development of the megasporocyte. The development of the embryo sac follows the Polygonum type. At 4–6 days after blooming, the embryos reached the globular stage, thereafter passing through the heart- and torpedo-shape stages before maturing into the cotyledon embryos. From blooming to seed maturity, it takes about 3–4 weeks under our conditions.

Adaptation of an ecdysone-based genetic switch for transgene expression in soybean seeds

Soybean was used as a model for studies of chemical induction of gene expression in seeds. A chimeric transcriptional activator, VGE, driven by the soybean seed glycinin G1 promoter, was used to induce the expression of an ER-targeted GFP(KDEL) reporter protein upon addition of the chemical ligand, methoxyfenozide. The chemical gene switch activated gene expression under in vitro conditions in somatic cotyledonary embryos and zygotic seed embryos cultured from transgenic soybean plants, as well as in seeds in planta under greenhouse conditions. The efficiency of induction of GFP expression under different growth conditions was strongly influenced by the developmental stage of the seed and availability of the inducer. The formation of ER-derived GFP-containing protein bodies in seed storage parenchyma cells was correlated with the level of induced expression.

Cyclic secondary somatic embryogenesis and efficient plant regeneration in camphor tree (Cinnamomum camphora L.)

An efficient protocol for secondary somatic embryogenesis in camphor tree is reported. Secondary somatic embryos (SSEs), initially obtained from the primary embryos of a nascent embryogenic culture in 2002, were proliferated and maintained for more than 4 yr via cyclic secondary somatic embryogenesis. Throughout this period, the embryo populations retained a high level of competence for plant regeneration. SSEs were produced on the surfaces of the cotyledons and radicular ends of maternal somatic embryos (MSEs). Histological observations of the various stages of secondary embryo development revealed four typical stages, namely, globular, heart-shaped, torpedo, and cotyledonary. The process of secondary embryogenesis continued in a cyclic way, with each newly formed embryo producing a subsequent generation of secondary embryos. In order to progress developmentally beyond proliferation cycles, cotyledonary embryos from one of embryogenic lines (L14) were cultured on Murashige and Skoog (MS) medium with 0.1–3.0 mg l−1 abscisic acid (ABA) or 0.05–1.0 mg l−1 thidiazuron (TDZ) in darkness for 2 mo to achieve maturation. Matured embryos were then transferred to MS-based germination medium containing either 0.1 mg l−1 TDZ, 0.2 mg l−1 indole-3-butyric acid (IBA), and 0.5 mg l−1 6-benzylaminopurine (BA) or 0.1 mg l−1 TDZ and 0.2 mg l−1 IBA and were cultured in light for germination. Over 50% of embryos matured in the presence of 0.5 mg l−1 ABA were able to germinate with shoots and poor root system. Frequencies of embryos germinating normal shoots among different genotypes did not change significantly. A total of 93% of the shoots from the germinated embryos converted to plantlets on half strength MS medium with 0.5 mg l−1 IBA by 3 wk. Plantlets acclimatized successfully to ex vitro conditions and developed as field-grown plants with normal appearance.

Simulating in ovulo osmotic potentials and O2 tensions normalize growth and pigmentation of immature cotton embryos

Oxygen tensions and osmotic potentials are important physiological factors of plant growth and development. To optimize these variables for cotton (Gossypium hirsutum L.) embryo culture, we quantified dissolved O2 (dO2) tensions, osmotic potentials, and pH at several locations in cotton ovules during embryony. Clark O2 microelectrodes were micromanipulated into intact ovules at an angle lateral to the developing embryo, and dO2 tensions were determined in integuments, nucelli and embryos. Ovular osmotic potentials and pH were determined from extracted ovule sap using vapor pressure osmometers and pH microelectrodes. Dissolved O2 tensions near or in embryos decreased from 104 mmol m−3 at 5 days post-anthesis (DPA) to 83 mmol m−3 at 18 DPA. Osmotic potentials of ovule sap decreased from −0.70 megapascals (MPa) at 2 DPA to −1.12 MPa at 8 DPA but then increased to −0.84 MPa by 17 DPA. Ovule sap pH at 5–17 DPA varied inconsistently and ranged from 5.4 to 6.5. Based on these results, a factorial experiment with two osmotic and three O2 treatments was designed. Immature embryos of cotton cultivar HS-26 were randomly assigned to the treatment combinations and cultured for 33 days. Oxygen treatments did not affect embryo growth, and there were no differences among treatments with regard to percentage of embryos that progressed to a more advanced stage of embryo development. However, cotyledons of embryos grown without osmotic adjustment were abnormally large, and embryos exposed to these treatments were abnormally brown. Browning was less severe for embryos exposed to low O2 tensions. Growth and pigmentation were most normal for embryos simultaneously exposed to O2 tensions and osmotic potentials that best simulated the observed in ovulo conditions.

Regeneration of soybean (Glycine max L. Merrill) through direct somatic embryogenesis from the immature embryonic shoot tip

We describe here a simple and efficient system of soybean (Glycine max L. Merrill) regeneration through direct somatic embryogenesis by using immature embryonic shoot tips (IEST) as explants. The cultivar Kaohsiung 10 (cv. K10) used in this study did not show embryogenic response either from mature seed-derived explants (cotyledon, embryonic tip, leaf, shoot and root) or immature cotyledons. However, it showed a high percentage (55.8%) of somatic embryo (SEm) formation from the IEST excised 2–3 wk after flowering, thus indicating the crucial roles of type and age of explants. The IEST put forth primary SEm after 2 mo of culturing on Murashige and Skoog (MS) medium supplemented with 6% sucrose, 164.8 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mM asparagine and 684 µM glutamine. Subsequently, secondary SEm were developed 1 mo after culturing on MS medium containing 123.6 µM 2,4-D and 3% sucrose. Cotyledonary embryos were induced on MS medium supplemented with 0.5% activated charcoal after 1 mo. The embryos were desiccated for 72–96 h on sterile Petri dishes and regenerated on hormone-free MS medium. Plantlets with well-developed shoots and roots were obtained within 5–6 mo of culturing of IEST. The SEm-derived plants were morphologically normal and fertile. Various parameters thought to be responsible for efficient regeneration of soybean through somatic embryogenesis are discussed. To our knowledge, this is the first report to employ IEST as explants for successful direct somatic embryogenesis in soybean.

Cytological analysis of hybrid embryos of intergeneric crosses between Salix viminalis and Populus species

Intergeneric hybridisation between Salix viminalis L. as the female and four Populus species (Populus trichocarpa, P. tremula, P. × canadensis and P. simonii) as male pollen donors was performed by in vitro stigma pollination. To overcome postzygotic barriers, transfer of hybrid embryos to new medium is necessary. We carried out detailed ultrastructural analyses to establish: (i) at which stage of embryo development the first signs of programmed cell death (PCD) could be detected; and (ii) at which stage the lack of serious or irreversible changes guaranteed that advanced development of hybrid plants could occur after embryo rescue. Transmission electron microscopy and confocal laser scanning microscopy revealed the presence of both developing and degenerating embryos. Developing globular, heart-shaped, and early cotyledonary embryos contained cells of correct ultrastructure. The only sign of intergeneric hybridisation was a delay in development for a few days, in comparison with control embryos. The earliest indicators of embryo degeneration were noted at 9 days after pollination (DAP). The most common indicators were excessive embryo vacuolisation, which was characterised by a large number of vesicles and formation of small vacuoles, as well as enlarged central vacuoles. Extended plastid thylakoids, folding of the cell wall, and autophagosomes were observed. Our detailed investigation of PCD in hybrid embryos enabled us to conclude that the embryo rescue technique was most effective in intergeneric willow × poplar crosses if applied between 9 and 16 DAP.

English

This study reports the induction of somatic embryos in Mucuna pruriens. Different explants cultured on MS medium supplemented with 11.31 iM 2,4-D produced golden yellow embryogenic callus that induced synchronized embryo development on MS basal liquid medium. Organization of pre-embryonic mass was noticed 15 d after sub culturing the callus, which progressively developed to globular, heart, torpedo and cotyledonary shaped embryos. Attempts to germinate them did not succeed as the cotyledonary embryos turned characteristic red and subsequently brown before finally turning black and unresponsive. The reaction was consistent across different media constituents, hormonal concentrations and pH ranges. Supplementation of anti-oxidants was also ineffective. Polyacrylamide gel electrophoretic analysis of the brown and non-brown embryos revealed quantitative differences in protein contents between the two. More detailed study is necessary to establish the precise cause for embryo browning and ways of its regulation.

Improved plantlet regeneration from open-pollinated families of Abies alba trees of Dobroč primeval forest and adjoing managed stand via somatic embryogenesis

AbstractPossibility to improve plantlet regeneration from Abies alba Mill. open-pollinated families of 4 trees in Dobroč primeval and 3 trees in managed forest was studied. Immature zygotic embryos were cultured in order to obtain initiation of embryogenic tissue. Totally, three from the families of the managed forest (57%) and two from the primeval families (50%) responded to initiation condition. Initiation frequencies among families ranged in managed forest: 4.5–56.2%, primeval: 5.4–16.8%. Maturation ability was shown by 77.3% of the primeval cell lines, 36.4% cell lines produced cotyledonary somatic embryos. In managed forest, in 62.5% of the cell lines embryo maturation was observed. Cotyledonary embryos developed only in 15% of cell lines. Regenerants were obtained from 9 cell lines of primeval and from 6 cell lines of managed forest. Biochemically, the mature somatic embryos were characterized by the variation in soluble and protein profiles. The corresponding profiles of insoluble proteins exhibited uniform pattern. The variation was characteristic for somatic embryos of individual cell lines rather than for the primeval and managed stands. Enzymatically, no indications were obtained supporting higher metabolic potential of somatic embryos derived from zygotic embryos of silver fir primeval stand than in somatic embryos originating from the trees of managed stand.

Abscisic acid and methyl jasmonate as regulators of ethylene biosynthesis during somatic embryogenesis of Medicago sativa L.

Effects of abscisic acid (ABA) and methyl jasmonate (MeJA) on ethylene production, ACC oxidase (ACO) activity, and content of 1-aminocyclopropane-1-carboxylic acid (ACC) during indirect somatic embryogenesis (SE) of Medicago sativa L. were studied. ABA and MeJA, at 50 μM, were applied during the induction, proliferation, or differentiation phase. ABA decreased ethylene production at the beginning of callus and SE induction and during the differentiation of somatic embryos. The hormone inhibited ACO activity in explants with overgrowing callus during the first two weeks of induction, in embryogenic suspension and also in differentiating embryos. The ACC content was reduced by ABA in callus at the end of SE induction, in embryogenic suspension and in globular embryos, but elevated in cotyledonary embryos. MeJA had no significant effect on ethylene production during M. sativa SE, despite the fact, that it inhibited ACO activity during the first two weeks of induction and in torpedo and cotyledonary embryos. The ACC content was increased by MeJA in 14-day-old callus and embryogenic suspension but was inhibited in globular embryos. Both ABA and MeJA seem to be involved in the regulation of ethylene biosynthesis during distinct phases of SE in M. sativa. It might be considered that exogenous ABA, more probably than MeJA, exerts its inhibitory effect on M. sativa somatic embryo formation by modifying ethylene production.

Polyamine profiles and biosynthesis in somatic embryo development and comparison of germinating somatic and zygotic embryos of Norway spruce

The polyamine (PA) contents and activities of PA biosynthetic enzymes in Norway spruce somatic embryos [Picea abies L. (Karst.), genotype AFO 541] were studied in relation to anatomical changes during their development, from proliferation to germination, and changes in these variables associated with the germination of mature somatic and zygotic embryos were compared. Activities of PA biosynthetic enzymes steadily increased during the development of somatic embryos, from embryogenic suspensor mass until early cotyledonary stages. In these stages, the spermidine (Spd) level was significantly higher than the putrescine (Put) level, and the increases coincided with the sharp increases in S-adenosylmethionine decarboxylase activity in the embryos. The biosynthetic enzyme activity subsequently declined in mature cotyledonary embryos, accompanied by sharp reductions in PA contents, especially in cellular Put contents in embryos from 6 weeks old through the desiccation phase (although the spermine level significantly increased during the desiccation phase), resulting in a shift in the Spd/Put ratio from ca. 2 in early cotyledonary embryos to around 10 after 3 weeks of desiccation. In mature zygotic embryos, Spd contents were twofold lower, but Put levels were higher, than in mature somatic embryos, hence their Spd/Put ratio was substantially lower (ca. 2, in both embryos and megagametophytes). In addition, the PA synthesis activity profiles in the embryos differed (ornithine decarboxylase and arginine decarboxylase activities predominating in mature somatic and zygotic embryos, respectively). The start of germination was associated with a rise in PA biosynthetic activity in the embryos of both origins, which was accompanied by a marked increase in Put contents in somatic embryos, resulting in the decline of Spd/Put ratio to about 2, similar to the ratio in mature and germinating zygotic embryos. The accumulation of high levels of PAs in somatic embryos may be causally linked to their lower germinability than in zygotic embryos.

Improving initiation, genotype capture, and family representation in somatic embryogenesis of Pinus radiata by a combination of zygotic embryo maturity, media, and explant preparation

The principal aim of this investigation was to improve somatic embryogenesis initiation and to enhance representation of families and genotypes within those families of Pinus radiata D. Don. A total of 19 open-pollinated seed families, many with unrelated and weakly related parents, were tested. Optimum stage of cone maturity for initiation success was tested by five collections made at 1 week intervals, spanning the developmental period from pro-embryo to cotyledonary embryos. Two media were compared; embryo-development media (EDM6) and a modified Litvay medium (Glitz). Two zygotic embryo explant-preparation techniques were tested; embryos with retained megagametophytes and excised embryos. Proliferating embryogenic tissues were obtained from all four treatments (2850 explants per treatment, 570 per collection time) for the 19 families. The best initiation rates were achieved with a combination of Glitz medium with excised zygotic embryos, with 55% of explants from all collections and all families combined giving rise to proliferating embryogenic tissue. At the optimal collection time for each of the families, this treatment gave a range of 47%–97% initiation success with an average of 70% per family.

Do stress-related phytohormones, abscisic acid and jasmonic acid play a role in the regulation of Medicago sativa L. somatic embryogenesis?

This study examined the role of endogenous abscisic acid (ABA) and jasmonic acid (JA) in indirect somatic embryogenesis of Medicago sativa L. A multiplex GC-MS/MS technique allowed quantitative single-run analyses of ABA, JA, 12-oxophytodienoic acid (OPDA) and indole-3-acetic acid (IAA). The preparation of initial explants led to a strong accumulation of ABA, JA and OPDA but not of IAA. Substantially higher levels of ABA, JA and OPDA were detected in developing somatic embryos than in callus or embryogenic suspension. Fluridone (FLD) decreased ABA, JA and OPDA levels. Indoprofen (INP) appeared to be a specific inhibitor of octadecanoid biosynthesis. Somatic embryo production and development were negatively affected by FLD or INP. Only INP (0.5 μM) applied during proliferation phase increased the number of cotyledonary embryos. The results strongly indicate the involvement of ABA and JA in somatic embryogenesis of M. sativa. Surprisingly, low IAA contents in comparison to stress-related compounds (ABA, JA and OPDA) were detected in explants, embryogenic tissues and somatic embryos.

FACTORS AFFECTING SOMATIC EMBRYOGENESIS INDUCTION AND DEVELOPMENT IN FEIJOA SELLOWIANA BERG.

Feijoa sellowiana Berg. (Myrtaceae) is a small tree known as feijoa or pineapple guava. In recent years, this species has gained horticultural and consumer recognition due to the qualities of its fruits which possess high levels of vitamin C and iodine. Somatic embryogenesis induction in feijoa was first achieved by our group in 1990 from mature zygotic embryos and, since then, several improvements have been made in the protocol for somatic embryo induction and conversion. However, some drawbacks still persist in the process of plant regeneration through somatic embryogenesis in feijoa such as the high number of anomalous somatic embryos formed, the low frequencies of plant somatic embryo conversion, and the reduced potential of adult explants to undergo somatic embryogenesis. Trying to overcome these problems several lines of investigation are being followed at our lab with the objective of improving the quality of the somatic embryos and, at the same time, to understand the factors responsible for these negative aspects. Recent experiments have shown that the culture of somatic embryos in abscisic acid for 4 weeks before conversion increased the levels of storage lipids and proteins in the cotyledons of the somatic embryos as well as the number of somatic embryos able to develop into plantlets. Moreover, it was found that somatic embryos of feijoa usually possess a morphologically variable suspensor-like structure that, in the first stages of development, is quite similar to the suspensor of the zygotic embryos. Another line of investigation indicated that an auxinic shock instead of a continuous culture in the presence of auxin reduced the levels of anomalous embryos formed. The role of the genotype on somatic embryo formation and some preliminary results about somatic embryogenesis induction from young leaves of adult micropropagated genotypes will also be presented.

Differential regulation of SERK, LEC1-Like and Pathogenesis-Related genes during indirect secondary somatic embryogenesis in grapevine

A culture model was developed in Vitis vinifera L., cultivar 'Chardonnay' for studying SE (Somatic Embryogenesis). The auxin 2,4-D (2,4-Dichlorophenoxyacetic acid) was used to induce indirect secondary embryogenesis at a high rate, starting from embryos derived from embryogenic cultures previously obtained. Cotyledonary embryos were shown to be more responsive to SE induction than embryos at the torpedo-stage and were used for molecular analyses. The expression of SERK (Somatic Embryogenesis Receptor Kinase), L1L (Leafy Cotyledon1 Like) and a set of PR (Pathogenesis-Related) genes was monitored during the whole SE process. VvSERK1, VvSERK2 and VvL1L were down-regulated by the 2,4-D treatment but expressed in embryonic tissues. On the contrary, VvPR1, VvPR8, VvPR10.1 and VvPR10.3 were strongly up-regulated by the 2,4-D treatment, and their transcripts were not or only weakly detected in clusters of secondary embryos. VvSERK3, VvPR3 and VvPR10.2 were more stably expressed in all tissues examined. The discussion deals with the putative role of the different genes in grapevine SE.

A gain-of-function mutation inIAA18altersArabidopsisembryonic apical patterning

Lateral organ emergence in plant embryos and meristems depends on spatially coordinated auxin transport and auxin response. Here, we report the gain-of-function iaa18-1 mutation in Arabidopsis, which stabilizes the Aux/IAA protein IAA18 and causes aberrant cotyledon placement in embryos. IAA18 was expressed in the apical domain of globular stage embryos, and in the shoot apical meristem and adaxial domain of cotyledons of heart stage embryos. Mutant globular embryos had asymmetric PIN1:GFP expression in the apical domain, indicating that IAA18-1 disrupts auxin transport. Genetic interactions among iaa18-1, loss-of-function mutations in ARF (Auxin response factor) genes and ARF-overexpressing constructs suggest that IAA18-1 inhibits activity of MP/ARF5 and other ARF proteins in the apical domain. The iaa18-1 mutation also increased the frequency of rootless seedlings in mutant backgrounds in which auxin regulation of basal pole development was affected. These results indicate that apical patterning requires Aux/IAA protein turnover, and that apical domain auxin response also influences root formation.

Reproductive characteristics of Opisthopappus taihangensis (Ling) Shih, an endangered Asteraceae species endemic to China

Opisthopappus taihangensis is an endangered species endemic to China and represents an important genetic resource for chrysanthemum improvement. We describe here its basic reproductive characteristics. The anthers are tetrasporangiate and the anther wall is composed of an epidermis, endothecium, middle layer and tapetum. The middle layer is lost by the microspore tetrad stage, and the tapetum disintegrates at the trinucleate pollen stage. Meiosis in the microspore mother cells is of the simultaneous type, and the tetrad is tetrahedral in shape. Mature pollen grains have three germinal apertures, two sperm nuclei and one vegetative nucleus. The in vitro pollen germination rate is only ∼10%. The ovule is anatropous, dual-integument, tenuinucellatae and the development of the embryo sac follows the Oenothera pattern. The archesporial cell below the nucellus epidermis functions as the megaspore mother cell and forms a linear tetrad. The embryo passes through a globular, heart and torpedo stage before maturing into a cotyledon embryo. The endangerment of O. taihangensis may be associated with low reproductive capacity, as a consequence of poor pollen viability.

Loblolly pine (Pinus taeda) female gametophyte and embryo pH changes during seed development

Stage-specific measurements of female gametophyte (FG) and embryo pH (hydrogen ion concentration) were made through the sequence of loblolly pine (Pinus taeda L.) seed development. The FG tissue from two open-pollinated trees showed similar pH profiles starting at 5.5 shortly after fertilization, increasing to about 6.1 at stage 7, levelling off at 6.3-6.5 towards the end of development and dropping to 6.0 just before cone opening. Measurements of the chalazal end were 0.05-0.2 pH units less than the micropylar end through early-to-mid-development. In contrast, embryo pH maintained a nearly constant value near 7.0 through development. Profiles of pH through seed development were similar whether portrayed by date or stage of embryo present in the seed. The pH profiles assisted in the development of improved embryogenic tissue initiation techniques. When post-autoclaving maturation medium pH was raised from about 5.3 in control medium to 5.7 or 5.5-5.7 with 2(n-morpholino)ethanesulphonic acid, cotyledonary embryo yields increased.

Asymmetric development of Cotyledons of Tomato Embryo: Testing the prediction of Self-Organization

AbstractDevelopmental biologists have long strived to understand how organisms acquire shape and form. The architecture of the mature plant is established during embryogenesis. They have learned much about how gene expression controls the specification of cell type and about how cells interact with one another to coordinate such specific decisions. Far less is known about autocatalytic feedback flow of resource molecules regulating a plant and its parts, shape and form. Indeed, it has even been proposed that the development of shape is not under genetic control but rather is determined by physical forces. Asymmetric development of sinks that depend on common resource pool has been viewed as a consequence of autocatalytic feedback process of flow of resource units into them. The feedback process implies that the stronger a sink is relative to its competitors, the greater is its probability of getting further resources as a non-linear function of its resource drawing ability and sink size. We have shown that this model contrasts with that of sink strength dependent model in its prediction of the subsequent development of the initial asymmetry of growing cotyledons of the tomato embryo (Lycopersicon esculentum L.), when their resource drawing ability is enhanced by exogenous application of the growth regulators (NAA, GA and BA), we test these prediction and show that the results are in conformity with the autocatalytic model proposed by Ganeshaiah and Uma Shaanker.

Asymmetric development of Cotyledons of Tomato Embryo: Testing the prediction of Self-Organization

Developmental biologists have long strived to understand how organisms acquire shape and form. The architecture of the mature plant is established during embryogenesis. They have learned much about how gene expression controls the specification of cell type and about how cells interact with one another to coordinate such specific decisions. Far less is known about autocatalytic feedback flow of resource molecules regulating a plant and its parts, shape and form. Indeed, it has even been proposed that the development of shape is not under genetic control but rather is determined by physical forces. Asymmetric development of sinks that depend on common resource pool has been viewed as a consequence of autocatalytic feedback process of flow of resource units into them. The feedback process implies that the stronger a sink is relative to its competitors, the greater is its probability of getting further resources as a non-linear function of its resource drawing ability and sink size. We have shown that this model contrasts with that of sink strength dependent model in its prediction of the subsequent development of the initial asymmetry of growing cotyledons of the tomato embryo (Lycopersicon esculentum L.), when their resource drawing ability is enhanced by exogenous application of the growth regulators (NAA, GA and BA), we test these prediction and show that the results are in conformity with the autocatalytic model proposed by Ganeshaiah and Uma Shaanker.

Seedless mechanism of a new mandarin cultivar ‘Wuzishatangju’ ( Citrus reticulata Blanco)

Seedlessness is often a desired characteristic for many types of fruits. A new seedless mandarin cultivar ‘Wuzishatangju’ and its original seeded cultivar ‘Shatangju’ were used to study the mechanism of seedlessness from the beginning of fertility. Embryonic development, compatibility of self-pollinated ‘Wuzishatangju’ and cross-pollinated ‘Wuzishatangju’ (‘Wuzishatangju’ × ‘Ponkan’, ‘Wuzishatangju’ × ‘Shatangju’) were studied. Results indicated that the male gametes of ‘Wuzishatangju’ were healthy. Fertility of the ‘Wuzishatangju’ embryo sac was normal and the mature embryo sac had one egg, two synergids, three antipodal cells and one big central cell containing two polar nuclei. Embryonic development of cross-pollinated ‘Wuzishatangju’ was normal. Many globular embryos and a few heart-shaped embryos were observed 2 weeks after cross-pollination (WACP). Heart- and torpedo-shaped embryos were observed at three WACP while embryos reached the torpedo stage at four WACP. Cotyledonary embryos were observed at five WACP and they developed into seeds while funiculi were still existent at seven WACP. However, embryonic development of self-pollinated ‘Wuzishatangju’ was abnormal and most embryos, which degenerated by 2 weeks after self-pollination (WASP), disappeared and seedless fruits were produced by four WASP. Pollen tubes of cross-pollinated ‘Wuzishatangju’ could grow normally in the stigma, style and ovary, and they entered into the embryo sac resulting in successful fertilization. The pollen tubes of self-pollinated ‘Wuzishatangju’ grew well in the stigma and style; however, when they entered into the ovary, they became twisted and could not enter the embryo sac. Results indicate that the activities of the male gamete and the fertility of the embryo sac of ‘Wuzishatangju’ were functioning normally with no embryo abortion during embryonic development. Gametophytic self-incompatibility (SI) caused seedlessness in ‘Wuzishatangju’ by blocking fertilization in the ovary.