Diploid Embryo Research Articles

A novel in vivo genome editing doubled haploid system for Zea mays L.

Doubled haploid (DH) technologies accelerate maize inbred development. Recently, methods using CRISPR-Cas have created gene-edited maize DH populations, albeit with relatively low editing frequencies. Restoring fertility via haploid chromosome doubling remains a critically important production constraint. Thus, improved editing and chromosome doubling outcomes are needed. Here we obtained maternally derived diploid embryos in vivo by ectopically co-expressing Zea mays BABY BOOM and cyclin D-like gene products within unfertilized egg cells. When combined with gene editing, the in vivo method enables the production of mature seed with a maternally derived, gene-edited diploid embryo without requiring in vitro tissue culture methods nor the use of a chemical chromosome doubling agent. In summary, we report a novel approach for creating gene-edited maize DH populations that we expect can accelerate genetic gain in a scalable, cost-effective manner.

P-805 Sperm and embryo genome editing by CRISPR-Cas9 in a mouse model

Abstract Study question We wonder whether it is possible to edit a specific gene in individual mammalian spermatozoa as well as embryo by using CRISPR-Cas9? Summary answer We were able to successfully edit the coat pigment gene on individual spermatozoa as well as embryos by injecting CRISPR-Cas9 during Piezo-ICSI. What is known already Earlier investigations into embryo genome editing with CRISPR-Cas9 were conducted during the S-phase or zygote stage, encountering obstacles such as mosaicism and loss of heterozygosity. To overcome these challenges, performing genome editing at the gamete level is considered more favorable. While it may be achievable in oocytes, sperm genome editing can be ambitious due to the tightly supercoiled DNA around the protamine cores. In initial attempts, simple permeabilization of the sperm membrane allowed CRISPR-Cas9 penetration into the cell; however, the attempts at genomic editing proved to be unsuccessful. Study design, size, duration In the past several months, oocytes were divided into two groups: half of them were used to perform embryo genome editing, whilst the other half was used to edit exclusively the sperm genome through oocyte-mediated sperm decondensation (OMSD), where a single spermatozoon was injected into an enucleated oocyte and used the ooplasmic machinery for DNA decondensation and editing. In both models, CRISPR-Cas9 was used to knock out Tyr gene to create an albino phenotype. Participants/materials, setting, methods B6D2F1 mice were used to retrieve oocytes and spermatozoa. A cohort of oocytes used for the OSMD approach were enucleated, whilst the others were maintained with an intact nucleus. All oocytes, enucleated or not, were injected with a single spermatozoon with Tyr-sgRNA and Cas9 protein. Embryos were cultured until the 8-cell stage and processed for T7E1 cleavage analysis. Genome editing efficiency was calculated following the manufacturer protocol. Main results and the role of chance Of the 40 oocytes used for the study, 20 intact oocytes were used to perform embryo genome editing in the standard fashion, while 20 were enucleated for OMSD experiments. After undergoing Piezo-ICSI with CRISPR-Cas9 solution, 90.0% (18/20) of intact oocytes fertilized. After 48 hours in culture, diploid embryos reached the 8-cell stage at 88.9% (16/18). The 584-bp region of the target site of extracted DNA was amplified and gene modification of the edited diploid embryo cohort was confirmed in 87.5% (14/16). Compared to the OMSD cohort, enucleated oocytes fertilized at 85.0% (17/20) while cleavage rate was lower at 64.7% (11/17) and sperm genome modification at 81.8% (9/11). Both diploid embryos and haploid androgenetic embryos evidence an editing efficiency of 36.8%. Therefore, we verified that genome editing on embryos and sperm achieved the same editing yield. Limitations, reasons for caution While the experimental observations are still limited, it is important to note that these results in the embryo model may be underestimated by the T7E1 cleavage assay that only recognize heterozygous modification. The technique needs further refinement to optimize targeting and editing efficiency. Wider implications of the findings Sperm genome decondensation through an OMSD approach allows CRISPR-Cas9 to edit the sperm genome in order to correct eventual pathogenic mutations present in the gamete. Moreover, these would be the only method to absolutely ensure uniform generation of resulting embryos. Trial registration number not applicable

P-142 The developmental time to reach the blastocyst stage in the human is an indicator of diploid mono-pronuclear embryos

Abstract Study question Is it possible to determine diploid mono-pronuclear (1PN) human embryos by the speed of development to blastocyst formation? Summary answer The developmental time to reach the blastocyst stage is a discrimination indicator for diploid 1PN embryos. What is known already We have previously reported that 80.7% of 1PN zygotes had a biparental chromosome using a Live-Cell imaging technique, and some of these developed to the blastocyst stage (Tokoro et al. ASRM 2013). Furthermore, we have reported that these blastocysts can result in a healthy live birth (Tsuji et al. ASRM 2020), and there was no effect on growth up to 5 years of age (Tsuji et al. ASRM 2023). On the other hand, it was also shown that a few of the 1PN embryos that developed into blastocysts were haploid embryos. Study design, size, duration This was a retrospective study that included 28 blastocysts derived from a 1PN zygote. The time period was 72 months (January 2017 to December 2022). These embryos are used for research following obtaining informed consent. Participants/materials, setting, methods The frequency rate of diploid embryos was compared from time of PN fading to blastocyst formation (Gardner’s classification: ≥ expansion grade 3). Two development time range groups were examined and designated: fast-developing embryos (65.7-78.7h) and slow-developing embryos (116.8-139.7h). Ploidy status was determined using whole blastocyst cells and NGS-based SNP analysis. Statistical significance was determined using Fisher’s exact test (level of P<0.05). Main results and the role of chance There was no significant difference in the average age (years) of the two development time groups (fast-developing embryos group; 35.5 +/- 5.5, slow-developing embryos group; 34.1 +/- 2.8). The frequency of diploid status in 1PN embryos with fast developing embryos was 100% (14/14), and in 1PN embryos designated as having slow development, the frequency of diploid embryos was only 14.3% (2/14), haploid embryos 71.4% (10/14), and other was 14.3% (2/14). As a result, the diploid embryo rate was significantly higher in fast-developing embryos than in slow-developing embryos (P<0.001). Limitations, reasons for caution This study was limited to extremely fast or slow-developing embryos to see trends. It is important to study the threshold time between diploidy and haploidy in 1PN embryos. Wider implications of the findings These results confirmed that fast developing 1PN embryos have a high diploid rate and can be used as transferable embryos in addition to 2PN embryos. Conversely, slow developing 1PN embryos should be considered to have a lower priority for transfer because of the high rate of haploid embryos. Trial registration number not applicable

P-563 Improved diagnosis in PGT-A samples with intermediate sex chromosomes copy number changes by adding ploidy and contamination analysis

Abstract Study question Does triple analysis of aneuploidy, ploidy and contamination improve the diagnosis in samples with intermediate copy number changes for the X and the Y chromosomes? Summary answer PGT-A with additional ploidy and contamination analysis improves the diagnosis of samples with intermediate sex chromosomes copy number changes, reducing non-informativity and the re-biopsy need. What is known already Next Generation Sequencing (NGS) in Preimplantation Genetic Testing for Aneuploidies (PGT-A) identifies chromosomal aneuploidies in trophectoderm (TE) biopsies. However, diagnosis limitations exist when the TE biopsy displays intermediate copy number changes for the X and the Y chromosomes, observed in ∼0.3% of biopsies. Few explanations are plausible, including mosaicism for the sex chromosomes, presence of exogenous DNA contamination or triploidy. A second TE biopsy (re-biopsy) could be recommended to obtain a conclusive result. However, additional Single Nucleotide Polymorphism (SNP) analysis to determine the ploidy status of the embryo and the presence of contamination in the biopsy can be an alternative. Study design, size, duration In this retrospective study, the impact on the diagnosis of SNPs analysis with a target panel was evaluated in PGT-A samples showing intermediate copy number changes for the X and the Y chromosomes. A total of 21 TE biopsies from January to December 2023 were included in the study. Participants/materials, setting, methods The TE biopsies were analysed using NGS with a proprietary protocol for library preparation, Ion Chef and Ion S5 System instruments for sequencing and a proprietary bioinformatics pipeline (v2.0) for 24-chromosomes aneuploidy testing. A re-biopsy was analysed in 8 embryos using the same protocol. Eighteen original biopsies were retrospectively evaluated for ploidy and contamination using an Ion AmpliSeq Custom Panel including SNPs (Thermo Fisher Scientific, USA) and a proprietary algorithm (v1.0) for data analysis. Main results and the role of chance The 21 TE biopsies displaying intermediate copy number changes for the X and the Y chromosomes were initially diagnosed as non-informative and candidates for re-biopsy to rescue diagnosis. A total of 8 embryos were re-biopsied (38.1%): 6 re-biopsies (75.0%) showed the same intermediate sex chromosomes copy number changes suggesting a triploid status of the embryo, 1 re-biopsy (12.5%) showed trisomy 22 suggesting DNA contamination in the original biopsy and, 1 re-biopsy (12.5%) displayed a mosaic monosomy X/XY suggesting a diploid-mosaic status of the embryo. In 18 original TE biopsies (85.7%), an additional ploidy and contamination analysis was done: 10 samples were triploid (55.6%), 6 samples showed DNA contamination (33.3%) and 2 samples were diploid (11.1%). In 5 embryos, the re-biopsy with the aneuploidy testing had been done: (i) in 4 triploid embryos the re-biopsies showed the same intermediate copy number changes for the sex chromosomes than the original biopsy, confirming the triploid status of the embryo; (ii) in 1 diploid embryo the re-biopsy showed a mosaic monosomy X/XY, confirming the diploid status and the sex chromosome mosaicism of the embryo. From the 21 embryos initially non-informative, only the 6 contaminated ones remained non-informative and candidates for re-biopsy (33.3%). Limitations, reasons for caution The limited number of samples with both analysis, ploidy/contamination by SNPs in the original sample and aneuploidy by NGS in the re-biopsy precluded to drawn stronger conclusions. This double-check would reinforce the benefit of doing additional ploidy/contamination analysis in these specific cases. Wider implications of the findings A high incidence of samples with intermediate sex chromosomes copy number are triploid, some have exogenous DNA contamination, and few are real mosaics for the sex chromosomes. Additional ploidy and contamination analysis could improve the PGT-A diagnosis and decrease mosaicism and non-informativity rates, reducing the embryos that would need re-biopsy. Trial registration number Not applicable

Two MADS-box proteins, AGL9 and AGL15, recruit the FIS-PRC2 complex to trigger the phase transition from endosperm proliferation to embryo development in Arabidopsis

Spatiotemporal regulation of gene expression by polycomb repressive complex 2 (PRC2) is critical for animal and plant development. The Arabidopsis fertilization independent seed (FIS)-PRC2 complex functions specifically during plant reproduction from gametogenesis to seed development. After a double fertilization event, triploid endosperm proliferates early, followed by the growth of a diploid embryo, which replaces the endosperm in Arabidopsis and many dicots. Key genes critical for endosperm proliferation such as IKU2 and MINI3 are activated after fertilization. Here we report that two MADS-box AGAMOUS-LIKE (AGL) proteins associate with the key endosperm proliferation loci and recruit the FIS-PRC2 repressive complex at 4–5 days after pollination (DAP). Interestingly, AGL9 and AGL15 only accumulate toward the end of endosperm proliferation at 4–5 DAP and promote the deposition of H3K27me3 marks at key endosperm proliferation loci. Disruption of AGL9 and AGL15 or overexpression of AGL9 or AGL15 significantly influence endosperm proliferation and cellularization. Genome-wide analysis with cleavage Under Targets and tagmentation (CUT&Tag) sequencing and RNA sequencing revealed the landscape of endosperm H3K27me3 marks and gene expression profiles in Col-0 and agl9 agl15. CUT&Tag qPCR also demonstrated the occupancy of the two MADS-box proteins and FIS-PRC2 on a few representative target loci. Our studies suggest that MADS-box proteins could potentially recruit PRC2 to regulate many other developmental processes in plants or even in fungi and animals.

Effectiveness of R1-nj Anthocyanin Marker in the Identification of In Vivo Induced Maize Haploid Embryos.

Doubled haploid (DH) technology has become integral to maize breeding programs to expedite inbred line development and increase the efficiency of breeding operations. Unlike many other plant species that use in vitro methods, DH production in maize uses a relatively simple and efficient in vivo haploid induction method. However, it takes two complete crop cycles for DH line generation, one for haploid induction and the other one for chromosome doubling and seed production. Rescuing in vivo induced haploid embryos has the potential to reduce the time for DH line development and improve the efficiency of DH line production. However, the identification of a few haploid embryos (~10%) resulting from an induction cross from the rest of the diploid embryos is a challenge. In this study, we demonstrated that an anthocyanin marker, namely R1-nj, which is integrated into most haploid inducers, can aid in distinguishing haploid and diploid embryos. Further, we tested conditions that enhance R1-nj anthocyanin marker expression in embryos and found that light and sucrose enhance anthocyanin expression, while phosphorous deprivation in the media had no affect. Validating the use of the R1-nj marker for haploid and diploid embryo identification using a gold standard classification based on visual differences among haploids and diploids for characteristics such as seedling vigor, erectness of leaves, tassel fertility, etc., indicated that the R1-nj marker could lead to significantly high false positives, necessitating the use of additional markers for increased accuracy and reliability of haploid embryo identification.

Influential factors and transcriptome analyses of immature diploid embryo anthocyanin accumulation in maize

BackgroundAnthocyanins are widely applied as a marker for haploid identification after haploid induction in maize. However, the factors affecting anthocyanin biosynthesis in immature embryos and the genes regulating this process remain unclear.ResultsIn this study, we analyzed the influence of genetic background of the male and female parents, embryo age and light exposure on anthocyanin accumulation in embryos. The results showed that light exposure was the most crucial factor enhancing the pigmentation of immature embryos. The identification accuracy of haploid embryos reached 96.4% after light exposure, but was only 11.0% following dark treatment. The total anthocyanin content was 7-fold higher in immature embryos cultured for 24 h under light conditions compared to embryos cultured in the dark. Transcriptome analysis revealed that the differentially expressed genes between immature embryos cultured for 24 h in dark and light chambers were significantly enriched in the pathways of flavonoid, flavone, flavonol and anthocyanin biosynthesis. Among the genes involved in anthocyanin biosynthesis, five up-regulated genes were identified: F3H, DFR, ANS, F3′H and the MYB transcription factor-encoding gene C1. The expression patterns of 14 selected genes were confirmed using quantitative reverse transcription-polymerase chain reaction.ConclusionLight is the most important factor facilitating anthocyanin accumulation in immature embryos. After 24 h of exposure to light, the expression levels of the structural genes F3H, DFR, ANS, F3′H and transcription factor gene C1 were significantly up-regulated. This study provides new insight into the factors and key genes regulating anthocyanin biosynthesis in immature embryos, and supports improved efficiency of immature haploid embryo selection during doubled haploid breeding of maize.

Polycomb-mediated repression of paternal chromosomes maintains haploid dosage in diploid embryos of Marchantia.

Complex mechanisms regulate gene dosage throughout eukaryotic life cycles. Mechanisms controlling gene dosage have been extensively studied in animals, however it is unknown how generalizable these mechanisms are to diverse eukaryotes. Here, we use the haploid plant Marchantia polymorpha to assess gene dosage control in its short-lived diploid embryo. We show that throughout embryogenesis, paternal chromosomes are repressed resulting in functional haploidy. The paternal genome is targeted for genomic imprinting by the Polycomb mark H3K27me3 starting at fertilization, rendering the maternal genome in control of embryogenesis. Maintaining haploid gene dosage by this new form of imprinting is essential for embryonic development. Our findings illustrate how haploid-dominant species can regulate gene dosage through paternal chromosome inactivation and initiates the exploration of the link between life cycle history and gene dosage in a broader range of organisms.

P–133 Monopronuclear (1PN) embryos can derive in healthy pregnancies

Abstract Study question Should 1PN embryos be considered suitable for transfer when normal development is observed at day 3 or day 5? Summary answer In IVF/ICSI cycles, 1PN zygotes are encountered in 2.7% of inseminated oocytes. Transfer of 1PN-embryos should be considered in the absence of suitable 2PN embryos. What is known already During in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) zygotes containing only a single pronucleus (monopronuclear, 1PN) are encountered in 1–7.7% of cases, while the display of two pronuclei is expected in a normally fertilised oocyte. A 1PN zygote can be of gynogenetic or androgenetic origin, but it can also be biparental. Gynogenetic and androgenetic 1PN embryos can be haploid or diploid, so a diploid 1PN embryo is not guaranteed to be normally fertilised. Generally, 1PN are discarded, as they have an increased risk for aneuploidy. However, sporadically they can develop into healthyl babies. Study design, size, duration 1PN-zygotes (n = 1287, 2.7% from all inseminated oocytes) from 1–1–2016 up to 15–12–2020 were retrospectively evaluated. The development and fate (discarded/transferred/cryopreserved) of all embryos were recorded. Embryos were evaluated at day 2, 3 or 5 of development. The policy of our unit is that, in absence of 2PN embryos, normal developed 1PN-embryos can be transferred on day 3. Supernumerary 1PN embryos can be cryopreserved at blastocyst stage. Ongoing pregnancies from fresh embryo transfers (ET) were analysed. Participants/materials, setting, methods In 946 IVF/ICSI cycles, at least one 1PN zygote was observed (total 1287 embryos). ICSI with ejaculated, PESA or TESE sperm counted for a total of 795 embryos, IVF cycles for 494 embryos. Embryo evaluation was performed using a home-made numerical algorithm: A (top embryo; 150–200 points), B (regular embryo; 100–149 points) or C (poor embryo; 0–99 points). Monopronuclear embryos always scored lower than equal developed 2PN embryos. Blastocyst evaluation was according to Gardner score. Main results and the role of chance From the 795 ICSI embryos, 49 (6.1%) were used for fresh ET (26 scored quality A or B), and a total of 60 embryos developed to blastocyst and were cryopreserved. From these 49 ICSI transfers, 4 (8.1%) ongoing pregnancies were obtained, all 4 from DET (1PN+2PN embryo), from which one twin pregnancy was confirmed. From the 494 IVF embryos, 41 (8.3%) were used for fresh ET (24 scored A or B), and 62 blastocysts were cryopreserved. A total of 9/41 (22%) ongoing pregnancies were obtained: 5 from SET (1PN) and 4 from DET (1PN+ 2PN embryo). Therefore, in only five IVF cycles a confirmed pregnancy was observed from a 1PN embryo (all A-quality embryos). Considering six ongoing pregnancies with complete certainty of monopronuclear origin from fresh tranfers could be confirmed from our retrospective data, we can conclude that although the live birth rate of these embryos is very low (around 0,5- 1.0%), they should not be discarded when development is normal and no dipronuclear embryos are present. Limitations, reasons for caution Cryo-thawing data is missing as these embryos were not differentially marked at freezing. Therefore, the cumulative pregnancies from monopronuclear embryos could be higher. Embryos were not evaluated in a time lapse system, so asynchronicity of PN formation could explain missing the right moment for evaluation, while normal fertilized. Wider implications of the findings: Notably, IVF monopronuclear embryos display a higher developmental potential than those derived from ICSI. We suggest that, in absence of dipronuclear embryos, culture to blastocyst stage before considering fresh ET or cryopreservation will help differentiate viable 1PN embryos, reducing the higher chance of genetic anomalies and miscarriages. Trial registration number N.A.

Sex-Specific Development in Haplodiploid Honeybee Is Controlled by the Female-Embryo-Specific Activation of Thousands of Intronic LncRNAs.

Embryonic development depends on a highly coordinated shift in transcription programs known as the maternal-to-zygotic transition (MZT). It remains unclear how haploid and diploid embryo coordinate their genomic activation and embryonic development during MZT in haplodiploid animals. Here, we applied a single-embryo RNA-seq approach to characterize the embryonic transcriptome dynamics in haploid males vs. diploid females of the haplodiploid insect honeybee (Apis mellifera). We observed typical zygotic genome activation (ZGA) occurred in three major waves specifically in female honeybee embryos; haploid genome activation was much weaker and occurred later. Strikingly, we also observed three waves of transcriptional activation for thousands of long non-coding transcripts (lncRNA), 73% of which are transcribed from intronic regions and 65% were specific to female honeybee embryos. These findings support a model in which introns encode thousands of lncRNAs that are expressed in a diploid-embryo-specific and ZGA-triggered manner that may have potential functions to regulate gene expression during early embryonic development in the haplodiploid insect honeybee.

Cross Inhibition of MPK10 and WRKY10 Participating in the Growth of Endosperm in Arabidopsis thaliana.

The product of double fertilization produces seed, which contains three components: triploid endosperm, diploid embryo, and maternal seed coat. Amongst them, the endosperm plays a crucial role in coordinating seed growth. Mitogen-activated protein kinase (MAPK) cascades are conserved in eukaryotes and involved in signal transduction of plant development. MPK3, MPK6, and MPK10 form a small group of MPKs family in Arabidopsis thaliana. MPK3 and MPK6 are extensively studied and were found to be involved in diverse processes including plant reproduction. However, less is known about the function of MPK10. Here, we found WRKY10/MINI3, a member of HAIKU (IKU) pathway engaging in endosperm development, and MPK10 is high-specifically expressed in the early developmental endosperm but with opposite gradients. We further proved that MPK10 and WRKY10 cross-inhibit the expression of each other. The inhibition effect of MPK10 on gene expression of WRKY10 and the downstream targets is supported by the fact that MPK10 interacts with WRKY10 and suppresses the transcriptional activity of WRKY10. Constantly, mpk10 mutants produce big seeds while WRKY10/MINI3 positively regulate seed growth. Altogether, our data provides a model of WRKY10 and MPK10 regulating endosperm development with a unique cross inhibitory mechanism.

Maize kernel development.

Maize (Zea mays) is a leading cereal crop in the world. The maize kernel is the storage organ and the harvest portion of this crop and is closely related to its yield and quality. The development of maize kernel is initiated by the double fertilization event, leading to the formation of a diploid embryo and a triploid endosperm. The embryo and endosperm are then undergone independent developmental programs, resulting in a mature maize kernel which is comprised of a persistent endosperm, a large embryo, and a maternal pericarp. Due to the well-characterized morphogenesis and powerful genetics, maize kernel has long been an excellent model for the study of cereal kernel development. In recent years, with the release of the maize reference genome and the development of new genomic technologies, there has been an explosive expansion of new knowledge for maize kernel development. In this review, we overviewed recent progress in the study of maize kernel development, with an emphasis on genetic mapping of kernel traits, transcriptome analysis during kernel development, functional gene cloning of kernel mutants, and genetic engineering of kernel traits.

Structure and histochemistry of sorghum caryopsis in relation to grain-filling

Anatomical and histochemical studies of ovary and caryopsis of sorghum reveal the importance of the chalazal complex in transporting nutrients from maternal sources to the filial diploid embryo and triploid endosperm. The presence of starch, protein, lipid, Ca, K, Mg, and Fe in various tissues at different stages of development can be revealed by a variety of histochemical techniques. Vascular supply ends at the base of the ovary and transport occurs through vascular parenchyma, pigment strand and nucellar projection where symplastic continuity is broken. Nutrients unloaded into an apoplastic placental sac then enter the endosperm and embryo through the aleurone transfer cells. The later possess characteristic wall ingrowth. The single layer of aleurone surrounding the endosperm may also help in transport during later stages of grain-filling. Grain-filling in C4 sorghum is compared with other C4 and C3 grasses showing the variety of strategies evolved to transport nutrients into filial tissues. Standardization of terminologies to describe the tissues of the crease region will help in further research and communication.

Morphological observations in durum wheat after embryo manipulations

In cereals, haploid or diploid embryo structures are suitable organisations in practice for amelioration or storage of genotypes or some other biotechnologies. It is possible to reveal of variation as different genotypes from heterozygote or landraces after making homozygote of recessive alleles by doubling of chromosomes using embryo rescue. In these genotypes, characters can be evaluated as qualitative or quantitatively, for instance; dark purple coleoptile could be observed in some doubled haploid (DH) genotypes. The amount of chlorophyll (a/b) and carotenoid have been evaluated in young leaves in DH-6 and DH-8 and some other durum wheat genotypes, while investigating their tolerances to salinity in laboratory conditions. Colour of grains (amount of carotenoid in harvested grains) in durum genotypes have also been evaluated with field experiment conditions in three different locations of Anatolia and also correlation (r) has been identified with other grain characters related with agronomical values. Here it is possible to determine the rate into correlation (path) of characters and direct and indirect effects of each other. Naturally, the relationships between amount of chlorophyl in young leaves and carotenoid in harvested grains and also harvest index (HI) of genotypes will be able to be interesting subjects to discuss.

Practical aspects on identification, cultivation and characteristics of varicella-zoster virus isolates

Until now, it has been considered that infectivity of the varicella-zoster virus (VZV) is closely related to target cell, and newly formed virus is not released into the culture medium. It is also known that it is hard to grow VZV in cell cultures, due to its slow replication rate and a limited range of sensitive cell cultures. In addition, VZV isolation depends on type of cell culture used, nature of clinical material, presence of viable virus and transport time. Objectives. To study production of infectious extracellular VZV in various cell cultures. Materials and methods. Eight cell cultures were used, including human embryonic diploid lung cells and human embryonic dermomuscular tissue (KM-27), as well as continuous human and monkey cell lines. Crusts detached from vesicular lesions were used as clinical isolates, which were placed into cryo-vials added with transport medium and transferred in liquid nitrogen. VZV infectivity was assessed in cell cultures by using hemo-adsorption assay with erythrocyte suspension isolated from guinea pig or human zero group blood and confirmed by indirect immunofluorescence with polyclonal sera from varicella or herpes zoster convalescents. Results. There were examined 27 clinical samples consisting of crusts from vesicular lesions isolated from patients with chickenpox, as well as one sample from 63-year old patient with exacerbated recurrent herpes zoster. Primary infection with clinical isolates was performed on diploid human lung embryo cells (HLEC) at low temperature. It was found that clinical samples collected within day 1–18 inclusive after the onset of skin eruption were able to induce cytopathic effects in HLEC cell monolayer such as cytolysis around dermal crusts. Specificity of cytopathic effect was confirmed by using real-time polymerase chain reaction (RT-PCR). Viral antigens were prepared on 7 cell lines infected with the laboratory strain Ellen VZV (USA) to assess the immune sera. A high anti-VZV specificity of mouse sera was detected by ELISA while all the lysates of infected cell lines were used as the solid-phase sorbent. In experiments on VZV reproduction demonstrated that extracellular virus was released into the culture medium starting from day 1 after infection of target cells, and infectivity of the virus-containing fluid ascends during further cultivation.

Maternal regulation of seed growth and patterning in flowering plants.

The plant haploid generation is specified late in higher plant development, and post-meiotic haploid plant cells divide mitotically to produce a haploid gametophyte, in which a subset of cells differentiates into the gametes. The immediate mother of the angiosperm seed is the female gametophyte, also called the embryo sac. In most flowering plants the embryo sac is comprised of two kinds of gametes (egg and central cell) and two kinds of subsidiary cells (antipodals and synergids) all of which descend from a single haploid spore produced by meiosis. The embryo sac develops within a specialized organ of the flower called the ovule, which supports and controls many steps in the development of both the embryo sac and the seed. Double fertilization of the central cell and egg cell by the two sperm cells of a pollen grain produce the endosperm and embryo of the seed, respectively. The endosperm and embryo develop under the influence of their precursor gametes and the surrounding tissues of the ovule and the gametophyte. The final size and pattern of the angiosperm seed then is the result of complex interactions across multiple tissues of three different generations (maternal sporophyte, maternal gametophyte, and the fertilization products) and three different ploidies (haploid gametophyte, diploid parental sporophyte and embryo, and triploid endosperm).

Evolution, Initiation, and Diversity in Early Plant Embryogenesis.

During embryogenesis in plants, cell identities are specified de novo, starting from a single cell. By combining imaging, genomic profiling, and genetics, principles of early plant development have been unraveled in the dicotyledonous plant Arabidopsis. A central emerging question, however, is how well zygotic embryogenesis in Arabidopsis reflects homologous processes in other plant species, including early diverging, non-flowering, and non-seed plants. Here, we consider plant embryogenesis with an emphasis on its evolutionary history, the diverse modes of its initiation, and the concepts in pattern formation among morphologically distinct plant groups. Furthermore, we explore challenges and future directions in plant embryogenesis research.

UPDATED PGDIS RECOMMENDATIONS FOR REPLACEMENT OF MOSAIC EMBRYOS

Objective To review current data on the developmental potential of mosaic embryos and update previous guidelines and recommendations for transfer of mosaic embryos. Background From PGT studies of blastocyst biopsies using next generation sequencing (NGS), the average frequency of embryonic mosaicism is around 10% (range 5% - 30%). Mosaicism is generated by mitotic nondisjunction errors that can affect any chromosome and manifests as either diploid mosaic, aneuploid mosaic or complex aneuploid mosaic embryos. The level of mosaicism, defined from analysis of a trophectoderm (TE) biopsy, is generally related to the timing of mitotic errors during preimplantation development. Currently, there are disputes about the biological relevance of mosaicism levels measured in a blastocyst TE biopsy by NGS. Thus, in situations where there is no available euploid embryo for transfer, even with current guidelines published by PGDIS, confusion still reigns in many clinics on whether to transfer diploid mosaic embryos. Recent observations A number of studies have now been performed to follow the outcome of transferring diploid mosaic embryos detected with either whole chromosome or segmental abnormalities involving one or more chromosomes. Compared to euploid embryos, diploid mosaics with 20-40% levels of trisomy or monosomy have slightly reduced implantation potential and, those that do successfully implant are capable of developing to live births without signs of chromosome disease syndromes. Re-analysis of mosaic embryos indicates that while the majority of second TE biopsies are concordant for the type of mosaicism originally detected by PGT, there can be differences in levels of mosaicism in the TE compartment, and in some cases, second biopsies appear euploid. In general, the mosaicism found in TE biopsies is not reflected in the ICM lineage. These studies suggest that the level of mosaicism determined by NGS and reported to the clinician from a PGT cycle may not always be indicative of the entire blastocyst. More recently, there is also growing evidence from sequencing data that artificial segmental mosaicism can occur due to biased chromosome representation in the WGA and library construction steps. Thus, biological and technical factors may occasionally falsely contribute to a diagnosis of embryonic mosaicism. Recommendations Clinics that routinely observe high levels of mosaicism should systemically review IVF and PGT protocols. In the absence of euploid embryos for transfer, genetic counseling should be first offered to explore the possibility of transferring a diploid mosaic embryo or initiating another PGT cycle. While outcomes are slightly compromised, diploid mosaic embryos with 20-50% mosaicism can be transferred regardless of the chromosomal abnormality involved. Transfer of embryos with 50-80% mosaicism should be considered with more caution, in particular, mosaic embryos associated with (i) trisomies of chromosome 21, 18 and 13 capable of developing to live births, (ii) trisomies causative of intrauterine growth restriction or (iii) segmentals causative of known chromosome disease syndromes. Following an ongoing pregnancy achieved with a diploid mosaic embryo, prenatal diagnosis by amniocentesis is highly recommended.

Maternal auxin supply contributes to early embryo patterning in Arabidopsis.

Introductory paragraphThe angiosperm seed is composed of three genetically distinct tissues: the diploid embryo that originates from the fertilized egg cell, the triploid endosperm that is produced from the fertilized central cell, and the maternal sporophytic integuments that develop into the seed coat1. At the onset of embryo development in Arabidopsis thaliana, the zygote divides asymmetrically producing a small apical embryonic cell, and a larger basal cell that connects the embryo to the maternal tissue2. The coordinated and synchronous development of the embryo and the surrounding integuments, and the alignment of their growth axes suggest communication between maternal tissues and the embryo. In contrast to animals, however, where a network of maternal factors that direct embryo patterning have been identified3,4, only a few maternal mutations have been described to affect embryo development in plants5–7. Early embryo patterning in Arabidopsis requires accumulation of the phytohormone auxin in the apical cell by directed transport from the suspensor8–10. However, the origin of this auxin has remained obscure. Here we investigate the source of auxin for early embryogenesis and provide evidence that the mother plant coordinates seed development by supplying auxin to the early embryo from the integuments of the ovule. We show that auxin response increases in ovules upon fertilization, due to upregulated auxin biosynthesis in the integuments, and this maternally-produced auxin is required for correct embryo development.

The long and winding road: transport pathways for amino acids in Arabidopsis seeds.

Pathways for assimilates. During their life cycle, plants alternate between a haploid stage, the gametophyte, and a diploid stage, the sporophyte. In higher plants, meiosis generates the gametophyte deeply embedded in the maternal tissue of the flower. The megaspore mother cell undergoes meiosis, and then, the surviving megaspore of the four megaspores produced undergoes mitotic divisions and finally gives rise to the female gametophyte, consisting of the egg cell, two synergids, the central cell, which due to the fusion of two nuclei is diploid (double haploid) in Arabidopsis and most angiosperms and the antipods, whose number is not fixed and varies significantly between species (Yadegari and Drews in Plant Cell 16(Suppl):S133-S141, 2004). The maternal tissues that harbor the female gametophyte and the female gametophyte are referred to as the ovule (Fig.1). Double fertilization of the egg cell and the central cell by the two generative nuclei of the pollen leads to the diploid embryo and the endosperm, respectively (Hamamura et al. in Curr Opin Plant Biol 15:70-77, 2012). Upon fertilization, the ovule is referred to as the seed. Seeds combine two purposes: to harbor storage compounds for use by the embryo upon germination and to protect the embryo until the correct conditions for germination are encountered. As a consequence, seeds are the plant tissue that is of highest nutritional value and the human diet, by a considerable amount, consists of seeds or seed-derived products. Amino acids are of special interest, because plants serve as the main source for the so-called essential amino acids, that animals cannot synthesize de novo and are therefore often a limiting factor for human growth and development (WHO in Protein and amino acid requirements in human nutrition. WHO technical report series, WHO, Geneva, 2007). The plant embryo needs amino acids for general protein synthesis, and additionally they are used to synthesize storage proteins in the seeds of certain plants, e.g., legumes as a resource to support the growth of the seedling after germination. The support of the embryo depends on transport processes that occur between the mother plant and the seed tissues including the embryo. In this review, we will focus on the processes of unloading amino acids from the phloem and their post-phloem transport. We will further highlight similarities between amino acid transport and the transport of the main assimilate and osmolyte, sucrose. Finally, we will discuss similarities and differences between different plant species in terms of structural aspects but for the molecular aspects we are almost exclusively focusing on Arabidopsis. Fig.1 Vascularization of the Arabidopsis ovule and seed. Plants expressing ER-localized mCherry under control of the companion cell-specific SUC2 promoter and ER-localized GFP under control of the sieve element marker PD1 as described (Müller et al. 2015) are shown to visualize the phloem in the funiculus and the chalazal regions. a Overview over an ovule. FG: female gametophyte. b A magnification of the region marked by a square in panel a. c Overview over a seed. ES: endosperm; E: embryo. d A magnification of the region marked by a square in panel c. The arrows in b and d point to the terminal companion cell and arrowheads to terminal sieve elements.