Development of polyspermic zygote and possible contribution of polyspermy to polyploid formation in angiosperms

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In angiosperms, polyploidization is a common phenomenon, and polyploidy would have played a major role in the long-term diversification and evolutionary success of plants. As for the mechanism of formation of autotetraploid plants, the triploid-bridge pathway, crossing between triploid and diploid plants, is considered as a major pathway. For the emergence of triploid plants, fusion of an unreduced gamete with a reduced gamete is generally accepted. In addition, the possibility of polyspermy has been proposed for maize, wheat and some orchids, although it has been regarded as an uncommon mechanism of triploid formation. One of the reasons why polyspermy is regarded as uncommon is because it is difficult to reproduce the polyspermy situation in zygotes and to analyze the developmental profiles of polyspermic triploid zygotes. Recently, polyspermic rice zygotes were successfully produced by electric fusion of an egg cell with two sperm cells, and their developmental profiles were monitored. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus in the polyspermic zygote, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle. The two-celled proembryos further developed and regenerated into triploid plants. These suggest that polyspermic plant zygotes have the potential to form triploid embryos, and that polyspermy in angiosperms might be a pathway for the formation of triploid plants.     

New advances and future directions in plant polyspermy

Plants have evolved a battery of mechanisms that potentially act as polyspermy barriers. Supernumerary sperm fusion to one egg cell has consequently long remained a hypothetical concept. The recent discovery that polyspermy in flowering plants is not lethal but generates viable triploid plants is a game changer affecting the field of developmental biology, evolution, and plant breeding. The establishment of protocols to artificially induce polyspermy together with the development of a high‐throughput assay to identify and trace polyspermic events in planta now provide powerful tools to unravel mechanisms of polyspermy regulation. These achievements are likely to open new avenues for animal polyspermy research as well, where forward genetic approaches are hampered by the fatal outcome of supernumerary sperm fusion.     

Abnormal fertilization is responsible for reduced fecundity following thiram-induced ovulatory delay in the rat

Brief exposure to some pesticides, applied during a sensitive window for the neural regulation of ovulation, will block the preovulatory surge of LH and, thus, delay ovulation. Previously, we have shown that a single i.p. injection of 50 mg/kg of thiram, a dithiocarbamate fungicide that decreases norepinephrine synthesis, on proestrus (1300 h) suppresses the LH surge and delays ovulation for 24 h without altering the number of oocytes released. However, when bred, the treated dams had a decreased litter size and increased postimplantation loss. We hypothesized that the reduced litter size in thiram-delayed rats was a consequence of altered oocyte function arising from intrafollicular oocyte aging. To test this hypothesis, we examined delayed oocytes, zygotes, and 2-cell embryos for evidence of fertilization and polyspermy. In addition, we used confocal laser-scanning microscopy to evaluate and characterize cortical granule localization in oocytes and release in zygotes, because the cortical granule response is a major factor in the normal block to polyspermy. Our results demonstrate that a thiram-induced, 24-h delay in ovulation alters the fertilizability of the released oocyte. Although no apparent morphological differences were observed in the unfertilized mature oocytes released following the thiram-induced delay, the changes observed following breeding include a significant decrease in the percentage of fertilized oocytes, a significant increase in polyspermic zygotes (21%), and a 10-fold increase in the number of supernumerary sperm in the perivitelline space. Importantly, all the polyspermic zygotes exhibited an abnormal pattern of cortical granule exudate, suggestive of a relationship between abnormal cortical reaction and the polyspermy in the delayed zygotes. Because polyspermy is associated with polyploidy, abnormal development, and early embryonic death, the observed polyspermy could explain the abnormal development and decreased litter size that we observed previously following thiram-delayed ovulation.     

Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.     

Timing of the first zygotic cleavage as a marker of developmental potential of mammalian embryos

Embryo quality related to its developmental potential is now one of the most important issues in modern embryology. It has been demonstrated that some in vitro produced blastocysts fail to hatch and implant after transfer despite a normal morphology. Although embryos are able to adjust to sub-optimal culture conditions, significant changes in expression profiles of developmentally important genes have been noticed. Timing of the first zygotic cleavage is considered a non-invasive marker of embryo developmental potential and has been successfully used in human IVF programs for identifying embryos of superior quality. Early-cleaving zygotes are more likely to develop to the blastocyst stage than their late-cleaving counterparts. The timing of the first zygotic cleavage has been associated with several parameters that may affect developmental potential of the resulting embryos. The mechanism causing variation in the timing of the first zygotic cleavage has not been identified. It may be related to culture environment or to some intrinsic factors within the oocyte, the sperm or both. In this paper we discuss some of the important aspects related to the timing of the first zygotic cleavage and its influence on the developmental competence of resulting embryos.     

In vitro production of embryos in swine.

In recent years, progress has been achieved in the production of pig embryos through IVM and IVF techniques. Cytoplasmic maturation of oocytes has been improved by modifications to IVM procedures. However, the historical problem of polyspermic penetration still remains a major issue to be solved. Recent studies indicate that the type of IVF medium and certain modifications to that medium can reduce polyspermy. Efforts should be directed to increase the developmental competence and quality of embryos. At present, many embryo culture (EC) media are available that can overcome the historical 4-cell block and support development of early in vivo derived embryos to the blastocyst stage. In contrast, blastocyst development of in vitro produced embryos in these culture media varies significantly. Furthermore, morphology and cell numbers in in vitro produced blastocysts are inferior to their in vivo counterparts. However, several modifications to EC techniques have improved embryo quality and developmental competence. Testing embryo viability through surgical transfer to recipient animals has resulted in acceptable pregnancy rates with moderate litter sizes. Although reliable in vitro systems are available for the generation of pig embryos, the problem of polyspermy and poor embryo development hamper their large-scale implementation. Further research efforts should be directed to improve oocyte/embryo quality and the methods to minimize polyspermy through development of novel IVM, IVF, and EC techniques.     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.     

Chromatin and microtubule morphology during the first cell cycle in bovine zygotes

Chromatin and microtubule configurations during the first cell cycle of bovine zygotes were analyzed by DNA staining and microtubule immunolocalization using an IVM/IVF system and oocytes matured and fertilized in vivo, in order to investigate the origin of the active centrosome and to characterize the nuclear and the cytoplasmic changes following bovine fertilization. Our results suggest that the paternal centrosome is active during early zygotic development, forming a conspicuous sperm aster soon after fertilization. We also report that polyspermy in bovine eggs, leads to the formation of numerous sperm asters with different degrees of association with the chromatin. The maternal structures in both monospermic and polyspermic zygotes can be lost or degenerate. Consequently, these cells may resume the first cell cycle as androgenotes, very often with several types of mitotic activity taking place in different regions of the cell cytoplasm at the same time. As indicated by a comparison of monospermic and polyspermic fertilization rates to rates of development, it is possible that some androgenetic embryos cleave and develop to the blastocyst stage. © 1993 Wiley-Liss, Inc.     

Diploid,but not haploid,human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells.     

Proteomic analysis of parthenogenetic and in vitro fertilized porcine embryos

Proteomic data from embryos are essential for the completion of whole proteome catalog due to embryo‐specific expression of certain proteins. In this study, using reverse phase LC‐MS/MS combined with 1‐D SDS‐PAGE, we identified 1625 mammalian and 735 Sus scrofa proteins from porcine zygotes that included both cytosolic and membranous proteins. We also found that the global protein profiles of parthenogenetically activated (PA) and in vitro fertilized (IVF) zygotes were similar but differences in expression of individual proteins were also evident. These differences were not due to culture conditions, polyspermy or non‐activation of oocytes, as the same culture method was used in both groups, the frequency of polyspermy was 24.3±3.0% and the rates of oocyte activation did not differ (p>0.05) between PA and IVF embryos. Consistent with proteomic data, fluorescent Hoechst 33 342 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay also revealed that PA embryos were of poor quality as they contained less cells per blastocyst and were more predisposed to apoptosis (p<0.05), although their in vitro development rates were similar. To our knowledge, this is the first report on global peptide sequencing and quantification of protein in PA and IVF embryos by LC‐MS/MS that may be useful as a reference map for future studies.     

In vitro development of polyspermic porcine oocytes: Relationship between early fragmentation and excessive number of penetrating spermatozoa

Embryo development during in vitro culture of polyspermic porcine oocytes was investigated in the present study. After in vitro fertilization (IVF) of in vitro matured oocytes, putative zygotes were centrifuged to visualize pronuclei. Two pronuclear (2PN) and poly-pronuclear (PPN) zygotes were selected and cultured in vitro. Their development to the blastocyst stage and total cell numbers, dead cell rates and ploidy at the blastocyst stage and morphology of resultant embryos after first cleavage were compared. A cleavage rate of PPN embryos was lower than that of 2PN (61.3% and 82.2%, respectively), however, the ability of cleaved embryos to develop to the blastocyst stage did not differ between the PPN and the 2PN groups (22.4% and 32.9%, respectively). Also there was no difference in total cell numbers and rates of dead cells between PPN and 2PN blastocysts. The majority of blastocysts in 2PN group were found to be diploid. In contrast, blastocysts in PPN group showed heterogeneous status in their ploidy including polyploidy and mixoploidy, whereas a remarkable proportion (31.3%) of them was found to be diploid. After the first cleavage (at 36 h after IVF), there was no difference in the number of nuclei/embryo between the two groups, nevertheless embryos in PPN group had significantly higher numbers of blastomeres than that of embryos in 2PN group, mainly due to an increased frequency of anuclear blastomeres. The present results indicate that correction of embryo ploidy in polyspermic embryos can occur during IVC. Nevertheless the frequency of partial fragmentation in polyspermic embryos is increased.     

Establishment of an in vitro fertilization system in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In vitro fertilization (IVF) systems using isolated male and female gametes have been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, as the female gametophytes of plants are deeply embedded within ovaries. In this study, a rice IVF system was established to take advantage of the abundant resources stemming from rice research for investigations into the mechanisms of fertilization and early embryogenesis. Fusion of gametes was performed using a modified electrofusion method, and the fusion product, a zygote, formed cell wall and an additional nucleolus. The zygote divided into a two-celled embryo 15–24 h after fusion, and developed into a globular-like embryo consisting of an average of 15–16 cells by 48 h after fusion. Comparison of the developmental processes of zygotes produced by IVF with those of zygotes generated in planta suggested that zygotes produced by IVF develop and grow into early globular stage embryos in a highly similar manner to those in planta. Although the IVF-produced globular embryos did not develop into late globular-stage or differentiated embryos, but into irregularly shaped cell masses, fertile plants were regenerated from the cell masses and the seeds harvested from these plants germinated normally. The rice IVF system reported here will be a powerful tool for studying the molecular mechanisms involved in the early embryogenesis of angiosperms and for making new cultivars.     

The human pronuclear ovum: Fine strcture of monospermic and polyspermic fertilization in vitro

The fine structure of pronuclear ova (monospermy and polyspermy) and one-cell embryos has been investigated in our IVF programme. Sixteen oocytes were collected at laparoscopy after appropriate hormonal stimulation and were matured and fertilized in vitro by methods that have given rise to normal pregnancies. Pronuclear ova showing monospermic fertilization had two vesicular pronuclei surrounded by aggregations of cellular organelles. The male pronucleus was closely associated with a sperm axoneme, while the female pronucleus was dismantling its envelope and condensing its chromatin ahead of its counterpart in late pronuclear ova. Each pronucleus had dispersed chromatin, dense compact nucleoli, and intranuclear annulate lamellae. Smooth endoplasmic reticulum, annulate lamellae, Golgi complexes, and mitochondria formed a conspicuous part of the perinuclear ooplasm. The one-cell embryos were either in syngamy or in the process of undergoing first cleavage. Positive evidence of cortical granule release and second polar bodies were detected in the perivitelline space. A block to polyspermy seemed to operate at the level of the inner zona. Dispermic and polyspermic ova had 3–16 pronuclei resembling those of monospermic ova and had sperm tails in the ooplasm. Sperm were also seen penetrating the inner zona and were occasionally found in the perivitelline space. Incomplete cortical granule release and early signs of cytoplasmic fragmentation were noted in polyspermic ova. Both normal and abnormal features of these ova are reported and compared with pronuclear structure in vivo and in vitro.     

In vitro fertilization: analysis of early post-fertilization development using cytological and molecular techniques

Methods have been developed which enable us to obtain in vitro fusion of pairs of sperm and egg cells, and sperm and central cells of angiosperms. Cultured products of such cell fusions develop progressively into zygotes, embryos and fertile plants, and endosperm, respectively. In vitro fusion of isolated gametes allows precisely timed examination of the earliest developmental processes following fertilization. When cultured, in vitro produced zygotes and primary endosperm cells organize themselves independently, and without any requirement for supporting tissues. This technology thus constitutes a unique model system for studies of early stages of zygotic embryogenesis and endosperm development. Following the adaptation of molecular techniques for use with only a few cells, it has proved possible to investigate developmental processes in these systems. This review describes the successful combination of molecular techniques with in vitro fertilization methods, and highlights results obtained with small numbers of reproductive cells isolated by microdissection.     

Regeneration of the barley zygote in ovule culture

An ovule culture technique has been established for barley that allows the regeneration of plants from zygotes. An average of 1.3 plantlets per ovule could be regenerated from more than 60% of the cultured ovules and about 75% of the regenerated plantlets developed into normal, fertile plants. The same regeneration frequencies were obtained in intact ovules and in ovules where the two integuments had been removed from the micropylar region. Unfertilized ovules and ovules where the fertilized eggs had been destroyed by a microinjection needle did not give rise to embryo-like structures. Plants could be regenerated from the zygote at the same frequency at developmental stages from immediately after fertilization until the formation of bicellular embryos. This tissue culture system appeared to be largely independent of genotype since similar regeneration frequencies were obtained in two different barley cultivars, Igri and Alexis, that in anther and microspore culture behave differently. The same technique has also been applied successfully in the wheat cultivar Walter.     

The potential significance of binovular follicles and binucleate giant oocytes for the development of genetic abnormalities

Normal development of a fertilizable female gamete emanates from a follicle containing only one oocyte that becomes haploid after first meiotic division. Binovular follicles including two oocytes and binucleate giant oocytes that are diploid after first meiosis constitute notable exceptions from this rule. Data provided by programmes of human-assisted reproduction on the occurrence of both phenomena have been reviewed to evaluate possible implications for the formation of genetic abnormalities. To exclude confusion with oocytes aspirated from two adjacent individual follicles, true binovularity has been defined as inclusion of two oocytes within a common zona pellucida or their fusion in the zonal region. A total of 18 conjoined oocytes have been reported and one of the oocyte was normally fertilized in seven cases. Simultaneous fertilization of both female gametes occurred only once. No pregnancy was achieved after transfer of an embryo from a binovular follicle. Binucleate giant oocytes have been observed sporadically but a few reports suggest an incidence of up to 0.3% of all gametes retrieved. Extensive studies performed by two independent centres demonstrated that giant oocytes are diploid at metaphase II, can undergo fertilization in vitro with formation of two or three pronuclei and develop into triploid zygotes and triploid or triploid/mosaic embryos. In summary, giant binucleate oocytes may be responsible for the development of digynic triploidy whereas the currently available data do not support a role of conjoined oocytes in producing dizygotic twins, mosaicism, chimaeras or tetraploidy. However, more information on the maturity and fertilizability of oocytes from binovular follicles is needed. Future studies should also evaluate a possible impact of pharmaceutical and environmental oestrogens on the formation of multiovular follicles.     

Experimentally produced diploid-triploid mouse chimaeras develop up to adulthood

Spontaneous diploid-triploid chimaeras occur sporadically in various mammalian species including man, but so far have never been produced experimentally. In order to get a deeper insight into the developmental consequences of this anomaly, we have developed two procedures that enabled for the first time to produce routinely diploid-triploid embryos, foetuses, and animals in the mouse. These procedures are: (1) aggregation of cleaving diploid embryos with triploid embryos produced by suppression of the second polar body in zygotes, and (2) fusion of a haploid karyoplast with one blastomere of the two-cell diploid embryos. The first procedure yielded 23 living and 6 dead postimplantation embryos and foetuses (age: 8th-19th day) out of which 22 were chimaeric. In addition, three chimaeric neonates reached adulthood. Two animals were fertile, and one--an overt chimaera--was an infertile male. The rate of postimplantation development of aggregation chimaeras was normal or only slightly retarded, and with one exception the foetuses were morphologically normal. Generally, the highest contribution of the 3n component in extra-embryonic structures was noted in the yolk sac, and usually it was higher than its contribution to the organs of the body. Chimaerism was most often noted in the liver, the heart, the intestine, and the lungs. Participation of triploid cells to all tissues studied, both in the body and in extra-embryonic structures, appeared to decrease slightly as development progressed. The second procedure yielded 10 foetuses and 6 adults. Three foetuses were chimaeric. Six fertile adults were probably non-chimaeras: the triploid component was absent in the coat and in the blood.     

Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification‐induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ‐H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage‐related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ‐H2AX foci in zygotes and two‐cell embryos. Trp53bp1 was upregulated in zygotes, two‐cell embryos and four‐cell embryos in the vitrified group, and Brca1 was increased in two‐cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two‐cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4′‐trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ‐H2AX foci in zygotes and two‐cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification‐induced abnormal ROS generation, γ‐H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ‐H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification‐associated γ‐H2AX accumulation is at least partially due to abnormal ROS generation.     

Pronuclear location before the first cell division determines ploidy of polyspermic pig embryos.

Polyspermy occurs frequently in the fertilization of mammalian eggs, but little is known about whether polyspermic eggs have developmental ability in vitro or in vivo. We previously reported that poly-pronuclear (PPN; 3 or more pronuclei) pig eggs developed normally to the blastocyst stage despite having fewer inner cell mass cell numbers as compared to blastocysts derived from two-pronuclear (2PN) eggs. Here it is shown that most PPN pig eggs have abnormal cleavage patterns (having 3 or more cells) in the first cell division and retarded development of pronuclei prior to syngamy as compared to 2PN eggs. Most blastocysts (14 of 18) that developed from PPN eggs showed abnormal ploidy (were haploid, triploid, and tetraploid) whereas 20 of 22 blastocysts derived from 2PN embryos were diploid. The size and morphology of most Day 40 fetuses that developed from PPN eggs appeared to be normal. Of 8 Day 40 fetuses analyzed, 1 was triploid (XXY) and another was a mosaic with both diploid (XX) and tetraploid cells (frequency of less than 10%, XXXX), and the others were diploid. Anomalies of chromosomal composition were not detected in these fetuses. Five live piglets and one dead piglet were born from two recipients of PPN eggs. It is proposed that not all pronuclei of PPN pig eggs participate in syngamy, resulting in diploid cells in the conceptus. Our data suggest that there are two types of pronuclei location in polyspermic pig eggs and that the resulting ploidy is determined at the zygote stage before the first cell division according to pronuclear location.     

Differentiation of isolated wheat zygotes into embryos and normal plants

Efficient and reproducible embryo development has been obtained from fertilized wheat (Triticum aestivum L.) egg cells isolated 3–6 h after hand-pollination of emasculated spikes. It is possible to routinely isolate viable zygotes from about 75% of the excised ovaries from cultivars of both winter and spring types. Co-culture with barley microspores which had been stimulated to sporophytic development resulted in embryonic development of the cultivated wheat zygotes. Within 23 h of pollination; the zygotes underwent their first cell division. They proceeded to develop into club-shaped embryos, most of which turned subsequently to dorsiventral differentiation. The morphological patterns of in-vitro-grown embryos were in accordance with those of normal zygotic embryos growing in planta. The formation of twin or multiple embryos originating from a single zygote was dependent on genotype and exogeneously supplied auxin. Upon transfer onto a suitable solidified medium, zygote-derived embryos usually germinated and developed into plants. After optimizing the feeder system, the nutrient medium and the concentration of 2,4-dichloro phenoxyacetic acid (2,4-D), more than 80 and 90% of the zygotes eventually developed into plants in genotypes Florida and Veery #5, respectively. All regenerated plants were morphologically normal and fertile. The in-vitro development from isolated zygotes of a higher-plant species into typically patterned zygotic embryos is shown here for the first time. Since the entire process, including early zygotic development, is now freely accessible to observation and micromanipulation, the method presented opens up new approaches in fundamental as well as applied fields of reproductive biology. Received: 4 September 1997 / Accepted: 28 November 1997