Whole-genome sequence analysis of mutations in rice plants regenerated from zygotes, mature embryos, and immature embryos.

Somaclonal variation was studied by whole-genome sequencing in rice plants (Oryza sativa L., 'Nipponbare') regenerated from the zygotes, mature embryos, and immature embryos of a single mother plant. The mother plant and its seed-propagated progeny were also sequenced. A total of 338 variants of the mother plant sequence were detected in the progeny, and mean values ranged from 9.0 of the seed-propagated plants to 37.4 of regenerants from mature embryos. The natural mutation rate of 1.2 × 10-8 calculated using the variants in the seed-propagated plants was consistent with the values reported previously. The ratio of single nucleotide variants (SNVs) among the variants in the seed-propagated plants was 91.1%, which is higher than 56.1% previously reported, and not significantly different from those in the regenerants. Overall, the ratio of transitions to transversions of SNVs was lower in the regenerants as shown previously. Plants regenerated from mature embryos had significantly more variants than different progeny types. Therefore, using zygotes and immature embryos can reduce somaclonal variation during the genetic manipulation of rice.

Transcriptome analyses uncover reliance of endosperm gene expression on Arabidopsis embryonic development.

Endosperm-embryo development in flowering plants is regulated coordinately by signal exchange during seed development. However, such a reciprocal control mechanism has not been clearly identified. In this study, we identified an endosperm-specific gene, LBD35, expressed in an embryonic development-dependent manner, by a comparative transcriptome and cytological analyses of double-fertilized and single-fertilized seeds prepared by using the kokopelli mutant, which frequently induces single fertilization events. Transcriptome analysis using LBD35 as a marker of the central cell fertilization event identified that 141 genes, including 31 genes for small cysteine-rich peptides, are expressed in a double fertilization-dependent manner. Our results reveal possible embryonic signals that regulate endosperm gene expression and provide a practicable method to identify genes involved in the communication during endosperm-embryo development.

Isolation of gametes and zygotes from Setaria viridis.

Setaria viridis, the wild ancestor of foxtail millet (Setaria italica), is an effective model plant for larger C4 crops because S. viridis has several desirable traits, such as short generation time, prolific seed production and a small genome size. These advantages are well suited for investigating molecular mechanisms in angiosperms, especially C4 crop species. Here, we report a procedure for isolating gametes and zygotes from S. viridis flowers. To isolate egg cells, ovaries were harvested from unpollinated mature flowers and cut transversely, which allowed direct access to the embryo sac. Thereafter, an egg cell was released from the cut end of the basal portion of the dissected ovary. To isolate sperm cells, pollen grains released from anthers were immersed in a mannitol solution, resulting in pollen-grain bursting, which released sperm cells. Additionally, S. viridis zygotes were successfully isolated from freshly pollinated flowers. Isolated zygotes cultured in a liquid medium developed into globular-like embryos and cell masses. Thus, isolated S. viridis gametes, zygotes and embryos are attainable for detailed observations and investigations of fertilization and developmental events in angiosperms.

Genome editing approaches using reproductive cells/tissues in flowering plants.

Targeted mutagenesis via programmable nucleases including the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system has been broadly utilized to generate genome-edited organisms including flowering plants. To date, specific expression of Cas9 protein and guide RNA (gRNA) in reproductive cells or tissues is considered one of the most effective genome-editing approaches for heritable targeted mutagenesis. In this report, we review recent advances in genome editing methods for reproductive cells or tissues, which have roles in transmitting genetic material to the next-generation, such as egg cells, pollen grains, zygotes, immature zygotic embryos, and shoot apical meristems (SAMs). Specific expression of Cas9 proteins in initiating cells efficiently induces targeted mutagenesis via Agrobacterium-mediated in planta transformation. In addition, genome editing by direct delivery of CRISPR/Cas9 components into pollen grains, zygotes, cells of embryos and SAMs has been successfully established to generate genome-edited plant lines. Notably, DNA-free genome editing by the delivery of Cas9-gRNA ribonucleoproteins (RNPs) is not associated with any legislative concerns about genetically modified organisms. In summary, the genome editing methods for reproductive cells or tissues have enormous potential for not only basic studies for plant reproduction but also applied sciences toward molecular plant breeding.

Regulatory functions of ROS dynamics via glutathione metabolism and glutathione peroxidase activity in developing rice zygote.

Reactive oxygen species (ROS) play essential roles in plant development and environmental stress responses. In this study, ROS dynamics, the glutathione redox status, the expression and subcellular localization of glutathione peroxidases (GPXs), and the effects of inhibitors of ROS-mediated metabolism were investigated along with fertilization and early zygotic embryogenesis in rice (Oryza sativa). Zygotes and early embryos exhibited developmental arrest upon inhibition of ROS production. Egg cells accumulated high ROS levels, and, after fertilization, intracellular ROS levels progressively declined in zygotes in which de novo expression of GPX1 and 3 was observed through upregulation of the genes. In addition to inhibition of GPX activity, depletion of glutathione impeded early embryonic development and led to failure of the zygote to appropriately decrease H2 O2 levels. Moreover, through monitoring of the glutathione redox status, the developing zygotes exhibited a progressive glutathione oxidation, which became extremely delayed under inhibited GPX activity. Our results provide insights into the importance of ROS dynamics, GPX antioxidant activity, and glutathione redox metabolism during zygotic/embryonic development.

Plant genome engineering from lab to field-a Keystone Symposia report.

Facing the challenges of the world's food sources posed by a growing global population and a warming climate will require improvements in plant breeding and technology. Enhancing crop resiliency and yield via genome engineering will undoubtedly be a key part of the solution. The advent of new tools, such as CRIPSR/Cas, has ushered in significant advances in plant genome engineering. However, several serious challenges remain in achieving this goal. Among them are efficient transformation and plant regeneration for most crop species, low frequency of some editing applications, and high attrition rates. On March 8 and 9, 2021, experts in plant genome engineering and breeding from academia and industry met virtually for the Keystone eSymposium "Plant Genome Engineering: From Lab to Field" to discuss advances in genome editing tools, plant transformation, plant breeding, and crop trait development, all vital for transferring the benefits of novel technologies to the field.

Effect of Paternal Genome Excess on the Developmental and Gene Expression Profiles of Polyspermic Zygotes in Rice.

Polyploid zygotes with a paternal gamete/genome excess exhibit arrested development, whereas polyploid zygotes with a maternal excess develop normally. These observations indicate that paternal and maternal genomes synergistically influence zygote development via distinct functions. In this study, to clarify how paternal genome excess affects zygotic development, the developmental and gene expression profiles of polyspermic rice zygotes were analyzed. The results indicated that polyspermic zygotes were mostly arrested at the one-cell stage after karyogamy had completed. Through comparison of transcriptomes between polyspermic zygotes and diploid zygotes, 36 and 43 genes with up-regulated and down-regulated expression levels, respectively, were identified in the polyspermic zygotes relative to the corresponding expression in the diploid zygotes. Notably, OsASGR-BBML1, which encodes an AP2 transcription factor possibly involved in initiating rice zygote development, was expressed at a much lower level in the polyspermic zygotes than in the diploid zygotes.

High-quality sugar production by osgcs1 rice.

Carbohydrates (sugars) are an essential energy-source for all life forms. They take a significant share of our daily consumption and are used for biofuel production as well. However, sugarcane and sugar beet are the only two crop plants which are used to produce sugar in significant amounts. Here, we have discovered and fine-tuned a phenomenon in rice which leads them to produce sugary-grain. We knocked-out GCS1 genes in rice by using CRISPR technology, which led to fertilization failure and pollen tube-dependent ovule enlargement morphology (POEM) phenomenon. Apparently, the POEMed-like rice ovule ('endosperm-focused') can grow near-normal seed-size unlike earlier observations in Arabidopsis in which gcs1 ovules ('embryo-focused') were aborted quite early. The POEMed-like rice ovules contained 10-20% sugar, with extremely high sucrose content (98%). Trancriptomic analysis revealed that the osgcs1 ovules had downregulation of starch biosynthetic genes, which would otherwise have converted sucrose to starch. Overall, this study shows that pollen tube content release is sufficient to trigger sucrose unloading at rice ovules. However, successful fertilization is indispensable to trigger sucrose-starch conversion. These findings are expected to pave the way for developing novel sugar producing crops suited for diverse climatic regions.

CRISPR/Cas9-Based Genome Editing Using Rice Zygotes.

Genome-editing technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Its application has been hindered in many cases, however, due to technical hurdles, such as the low rate of macromolecule delivery into plant cells and tissues or difficulties in plant transformation and regeneration. Here, a protocol for CRISPR/Cas9-based genome editing using rice zygotes is described. The genome-editing system is constructed via polyethylene glycol/calcium-mediated transfection with CRISPR/Cas9 components in rice zygotes, which are produced by in vitro fertilization of isolated rice gametes. Plasmid DNA harboring a CRISPR/Cas9 expression cassette or preassembled Cas9 protein-guide RNA ribonucleoproteins is transfected into zygotes, resulting in the regeneration of plants with a high frequency of the targeted mutation, which is either mono-allelic or bi-allelic, in the range of about 4% to 64%. Application of the present method has the potential to advance the molecular breeding of other crop species as well as rice. © 2020 Wiley Periodicals LLC.

In Vitro Production of Zygotes by Electrofusion of Rice Gametes.

In angiosperms, fertilization and embryogenesis occur in the embryo sac, which is deeply embedded in ovular tissue. In vitro fertilization (IVF) systems using isolated gametes have been utilized to dissect postfertilization events in angiosperms, such as egg activation, zygotic development, and early embryogenesis. In addition, using IVF systems, interspecific zygotes and polyploid zygotes have been artificially produced, and their developmental profiles/mechanisms have been analyzed. Taken together, the IVF system can be considered a powerful technique for investigating the fertilization-induced developmental sequences in zygotes and generating new cultivars with desirable characteristics. Here, we describe the procedures for the isolation of rice gametes, electrofusion of gametes, and the culture of the produced zygotes and embryo.

Polyspermy in angiosperms: Its contribution to polyploid formation and speciation.

Polyploidization has played a major role in the long-term diversification and evolutionary success of angiosperms. Triploid formation among diploid plants, which is generally considered to be achieved by fertilization of an unreduced gamete with a reduced one, has been accepted as a means of polyploid production. In addition, it has been supposed that polyspermy also contributes to the triploid formation in maize, wheat, and some orchids; however, such a mechanism has been considered uncommon because reproducing the polyspermic situation and unambiguously investigating developmental profiles of polyspermic zygotes are difficult. To overcome these problems, rice polyspermic zygotes have been successfully produced by electrofusion of an egg cell with two sperm cells, and their developmental profiles have been monitored. The triploid zygotes progress through karyogamy and divide into two-celled embryos via a typical bipolar mitotic division; the two-celled embryos further develop into triploid plants, indicating that polyspermic plant zygotes, unlike those of animals, can develop normally. Furthermore, progenies consisting of triparental genetic materials have been successfully obtained in Arabidopsis through the pollination of two different kinds of male parents with a female parent. These different pieces of evidence for development and emergence of polyspermic zygotes in vitro and in planta suggest that polyspermy is a key event in polyploidization and species diversification.

Gene Expression and Genome Editing Systems by Direct Delivery of Macromolecules Into Rice Egg Cells and Zygotes.

Polyethylene glycol calcium (PEG-Ca2+)-mediated transfection allows rapid and efficient examination to analyze diverse cellular functions of genes of interest. In plant cells, macromolecules, such as DNA, RNA and protein, are delivered into protoplasts derived from somatic tissues or calli via PEG-Ca2+ transfection. To broaden and develop the scope of investigations using plant gametes and zygotes, a procedure for direct delivery of macromolecules into these cells has recently been established using PEG-Ca2+ transfection. This PEG-Ca2+-mediated delivery into rice egg cells/zygotes consists of four microtechniques, (i) isolation of gametes, (ii) production of zygotes by electrofusion of gametes, (iii) PEG-Ca2+-mediated delivery of macromolecules into isolated egg cells or produced zygotes, and (iv) culture and subsequent analyses of the transfected egg cells/zygotes. Because the full protocol for microtechniques (i) and (ii) have already been reported in Toda et al., 2016 , microtechniques (iii) and (iv) are mainly described in this protocol.

An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice.

Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants1,2. Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9-guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment3, CRISPR-Cas9 integration increases the chance of off-target modifications4, and foreign DNA sequences cause legislative concerns about genetically modified organisms5. Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol-calcium (PEG-Ca2+)-mediated transfection in tobacco, Arabidopsis, lettuce, rice6, Petunia7, grapevine, apple8 and potato9, or into embryo cells by biolistic bombardment in maize10 and wheat11. However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9-gRNA RNPs into plant zygotes. Cas9-gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes12 and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14-64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species.

Expression of Genes from Paternal Alleles in Rice Zygotes and Involvement of OsASGR-BBML1 in Initiation of Zygotic Development.

Upon fertilization in angiosperms, one sperm cell fuses with the egg cell to produce a zygote, and, via karyogamy, the parental genetic information is combined to form the diploid zygotic genome. Recently, analyses with parentally imbalanced rice zygotes indicated that parental genomes are utilized synergistically in zygotes with different functions, and that genes transcribed from the paternal or maternal allele might play important roles in zygotic development. Herein, we first conducted single nucleotide polymorphism-based mRNA-sequencing using intersubspecific rice zygotes. Twenty-three genes, with paternal allele-specific expression in zygotes, were identified, and, surprisingly, their allele dependencies in the globular-like embryo tended to be biallelic. This suggests that the paternal-dependent expression of these genes is temporary, occurring during the early stages of zygote development. Of the 23 genes, we focused on Oryza sativa Apospory-specific Genome Region (ASGR)-BABY-BOOM LIKE (BBML) 1 (OsASGR-BBML1), presumed to encode an AP2-transcription factor, due to its reported role in zygotic development. Interestingly, ectopic expression of OsASGR-BBML1 in egg cells induced nuclear and cell divisions, indicating that exogenously expressed OsASGR-BBML1 converts the proliferation status of the egg cell from quiescent to active. In addition, the suppression of the function of OsASGR-BBML1 and its homologs in zygotes resulted in the developmental arrest, suggesting that OsASGR-BBML1 possesses an important role in initiating zygotic development. Monoallelic or preferential gene expression from the paternal genome in the zygote might be a safety mechanism allowing egg cells to suppress the gene expression cascade toward early embryogenesis that is normally triggered by fusion with a sperm cell.

An imbalanced parental genome ratio affects the development of rice zygotes.

Upon double fertilization, one sperm cell fuses with the egg cell to form a zygote with a 1:1 maternal-to-paternal genome ratio (1m:1p), and another sperm cell fuses with the central cell to form a triploid primary endosperm cell with a 2m:1p ratio, resulting in formation of the embryo and the endosperm, respectively. The endosperm is known to be considerably sensitive to the ratio of the parental genomes. However, the effect of an imbalance of the parental genomes on zygotic development and embryogenesis has not been well studied, because it is difficult to reproduce the parental genome-imbalanced situation in zygotes and to monitor the developmental profile of zygotes without external effects from the endosperm. In this study, we produced polyploid zygotes with an imbalanced parental genome ratio by electro-fusion of isolated rice gametes and observed their developmental profiles. Polyploid zygotes with an excess maternal gamete/genome developed normally, whereas approximately half to three-quarters of polyploid zygotes with a paternal excess showed developmental arrests. These results indicate that paternal and maternal genomes synergistically serve zygote development with distinct functions, and that genes with monoallelic expression play important roles during zygotic development and embryogenesis.

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.

Development of gene expression system in egg cells and zygotes isolated from rice and maize.

Polyethylene glycol calcium (PEG-Ca2+) transfection-mediated analysis allows rapid and efficient examination of gene function. To investigate the diverse cellular functions of genes of interest in plant cells, macromolecules, such as DNA, RNA, and proteins, are delivered into protoplasts prepared from somatic tissues or calli using a PEG-Ca2+ transfection procedure. To take advantage of this macromolecule delivery system in the reproductive and developmental biology of angiosperms, this study established a PEG-Ca2+ transfection system with isolated egg cells and zygotes. The conditions for PEG and plasmid DNA concentrations for transfection of rice egg cells were first addressed, and ~30% of PEG-Ca2+-transfected egg cells showed exogenous and transient expressions of fluorescent proteins from plasmid DNA delivered into the cells. Interestingly, a dual expression of two different fluorescent proteins in the same egg cell using two kinds of plasmid DNAs was also observed. For PEG-Ca2+ transfection with maize zygotes, ~80% of zygotes showed expression of GFP proteins from plasmid DNA. Importantly, PEG-transfected zygotes developed normally into cell masses and mature plants. These results suggest that the present PEG-Ca2+-mediated transient expression system provides a novel and effective platform for expressing and analyzing genes of interest in egg cells and zygotes. Moreover, combined with the CRISPR/Cas9 approach, the present transient expression system in zygotes will become a powerful and alternative tool for the preparation of gene-edited plants.

Formation of triploid plants via possible polyspermy.

Polyploidization is a common phenomenon in angiosperms, and polyploidy has played a major role in the long-term diversification and evolutionary success of plants. Triploid plants are considered as the intermediate stage in the formation of stable autotetraploid plants, and this pathway of tetraploid formation is known as the triploid bridge. As for the mechanism of triploid formation among diploid populations, 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 polyploid 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 zygotes. In the study, we produced polyspermic rice zygotes by electric fusion of an egg cell with two sperm cells and monitored their developmental profiles. The two sperm nuclei and the 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 developed and regenerated into triploid plants. These results 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.

Development of Polyspermic Rice Zygotes.

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In most animals and fucoid algae, polyspermy block occurs at the plasmogamy step. Because the polyspermy barrier in animals and in fucoid algae is incomplete, polyspermic zygotes are generated by multiple fertilization events. However, these polyspermic zygotes with extra centrioles from multiple sperms show aberrant nuclear and cell division. In angiosperms, polyspermy block functions in the egg cell and the central cell to promote faithful double fertilization, although the mechanism of polyspermy block remains unclear. In contrast to the case in animals and fucoid algae, polyspermic zygotes formed in angiosperms are not expected to die because angiosperms lack centrosomes. However, there have been no reports on the developmental profiles of polyspermic zygotes at cellular level in angiosperms. In this study, we produced polyspermic rice zygotes by electric fusion of an egg cell with two sperm cells, and monitored their developmental profiles. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle, as observed during mitosis of a diploid zygote. The two-celled proembryos further developed and regenerated into triploid plants. These findings suggest that polyspermic plant zygotes have the potential to form triploid embryos. Polyspermy in angiosperms might be a pathway for the formation of triploid plants, which can contribute significantly to the formation of autopolyploids.