Pollen Mother Cells Research Articles

Cytological and morphology characteristics of natural microsporogenesis within Camellia oleifera.

The online version contains supplementary material available at 10.1007/s12298-021-01002-5.

The impaired biosynthetic networks in defective tapetum lead to male sterility in watermelon

Heterosis has been widely applied in watermelon breeding, because of the higher resistance and yield of hybrid. As the basis of heterosis utilization, genic male sterility (GMS) is an important tool for facilitating hybrid seed production, while the detailed mechanism in watermelon is still largely unknown. Here, we report a spontaneous mutant Se18 exhibited complete male sterility due to the uniquely multilayered tapetum and the un-meiotic pollen mother cells during pollen development. Using TMT based quantitative proteomic analyses, a total of 348 differentially abundant proteins (DAPs) were detected with the overwhelming majority down-regulated in mutant Se18. By analyzing the putative orthologs/homologs of Arabidopsis GMS related genes, the biosynthesis and transport of sporopollenin and tryphine precursors were predictably altered in mutant compared to its sibling wild type. Moreover, the general phenylpropanoid pathway as well as its related metabolisms was also expectably impaired in mutant, coincident with the pale yellow petals. Notably, some key transcriptional factors regulating tapetum development, together with their down-regulated targets, offered potentially valuable candidates regarding of male sterility. Collectively, the disrupted regulatory networks underlying male sterility of watermelon was proposed, which provide novel insights into genetic mechanism of male reproductive process and rich gene resources for future research. SignificanceWatermelon is an importantly economical cucurbit crop worldwide, with high nutritional value. Although several male sterile mutants have been identified in watermelon, the underlying molecular mechanism is poorly elucidated. Comparative cytological analysis revealed that the defective development of tapetum was responsible for male sterility in mutant Se18. Combined with the morphological comparison, male floral buds at 2.0–2.5 mm in diameter were confirmed with no obvious phenotypic differences but distinct cytological defects, which were in turn sampled for TMT based proteomic analyses. Referring to functionally characterized GMS related genes, the genetic pathway DYT1-TDF1-AMS-MS188-MS1 regulating tapetum development, together with some downstream targets, were considerably altered in mutant Se18. Moreover, enrichment analyses illustrated the general phenylpropanoid related metabolisms, as well as the biosynthesis and transport of sporopollenin and tryphine precursors, were significantly disrupted in defective anther development. Collectively, the proposed regulatory networks in watermelon not only contribute to a better understanding of molecular mechanisms underlying male sterility, but also provide valuable GMS related candidates for future researches.

Male meiosis and pollen morphology in diploid Indonesian wild bananas and cultivars

Breeding of banana is hampered by its genetic complexity, structural chromosome rearrangements and different ploidy levels. Various scientific disciplines, including cytogenetics, linkage mapping, and bioinformatics, are helpful tools in characterising cultivars and wild relatives used in crossing programs. Chromosome analysis still plays a pivotal role in studying hybrid sterility and structural and numerical variants. In this study, we describe the optimisation of the chromosome spreading protocol of pollen mother cells focusing on the effects of standard fixation methods, duration of the pectolytic enzyme treatment and advantages of fluorescence microscopy of DAPI stained cell spreads. We demonstrate the benefits of this protocol on meiotic features of five wild diploid Musa acuminata bananas and a diploid (AA) cultivar banana “Rejang”, with particular attention on pairing configurations and chromosome transmission that may be indicative for translocations and inversions. Pollen slides demonstrate regular-shaped spores except “Rejang”, which shows fertile pollen grains of different size and sterile pollen grains, suggesting partial sterility and unreduced gamete formation that likely resulted from restitutional meiotic divisions.

Characterization of fertility alteration and marker validation for male sterility genes in novel PTGMS lines hybrid rice.

Photoperiod and thermosensitive genetic male sterile (PTGMS) lines have become one of the main sources of global rice production increasing. This study was conducted to evaluate the fertility alteration and validate the male sterility genes using validation markers in novel Egyptian Indica and Japonica PTGMS lines under natural conditions. The study revealed that the new genetic male sterile lines belong to the type of photo–thermosensitive genetic male sterility (PTGMS). The fertility alteration of these lines has influenced by photoperiod and temperature interaction. The new PTGMS lines have three sensitive periods of fertility alteration; transformation, sterility, and fertility period. Furthermore, the sensitive stage of fertility transformation might be from secondary branch primordial to pollen mother cells (PMC) meiosis. Under the natural Sakha condition, the new PTGMS lines were stable sterile under the condition of day length upper 13,75 h and temperature over 25 °C, while its convert to fertile under day length under 13 h, and temperature lower than 24 °C. The co-dominant markers identified the pms3 and tms5 genes in the new PTGMS lines, indicated that the fertility alteration in these lines controlled by photoperiod and thermosensitive stages.

Chromosome behavior during meiosis in pollen mother cells from Saccharum officinarum \xd7 Erianthus arundinaceus F1 hybrids

BackgroundIn recent years, sugarcane has attracted increasing attention as an energy crop. Wild resources are widely used to improve the narrow genetic base of sugarcane. However, the infertility of F1 hybrids between Saccharum officinarum (S. officinarum) and Erianthus arundinaceus (E. arundinaceus) has hindered sugarcane breeding efforts. To discover the cause of this infertility, we studied the hybridization process from a cytological perspective.ResultsWe examined the meiotic process of pollen mother cells (PMCs) in three F1 hybrids between S. officinarum and E. arundinaceus. Cytological analysis showed that the male parents, Hainan 92–77 and Hainan 92–105, had normal meiosis. However, the meiosis process in F1 hybrids showed various abnormal phenomena, including lagging chromosomes, micronuclei, uneven segregation, chromosome bridges, and inability to form cell plates. Genomic in situ hybridization (GISH) showed unequal chromatin distribution during cell division. Interestingly, 96.70% of lagging chromosomes were from E. arundinaceus. Furthermore, fluorescence in situ hybridization (FISH) was performed using 45S rDNA and 5S rDNA as probes. Either 45S rDNA or 5S rDNA sites were lost during abnormal meiosis, and results of unequal chromosomal separation were also clearly observed in tetrads.ConclusionsUsing cytogenetic analysis, a large number of meiotic abnormalities were observed in F1. GISH further confirmed that 96.70% of the lagging chromosomes were from E. arundinaceus. Chromosome loss was found by further investigation of repeat sequences. Our findings provide insight into sugarcane chromosome inheritance to aid innovation and utilization in sugarcane germplasm resources.

Colchicine induction of 'Old Blush' 2n pollen for the hybridization and breeding of tetraploid rose.

Obtaining 2n pollen from the diploid Chinese old rose ‘Old Blush’ through artificial induction is one important means of hybridizing and breeding modern tetraploid roses. We used colchicine-induced 2n pollen to assess normal viability during hybridization and fructification. The results showed that the pollen mother cell had lagging chromosomes and parallel spindles at meiosis I stage, following which the 2n pollen was produced from dyads and triads with doubled chromosomes. We obtained 4.30% viable 2n pollen, which was significantly higher than the yield of the spontaneous 2n pollen (1.00%) using an optimal treatment combination of induction for 24 h with 0.50% colchicine. There was no significant difference between the external morphology of the induced 2n pollen and the spontaneous 2n pollen, whereas both types of 2n pollen possessed finer furrows, and fewer and smaller pores than the 1n pollen, and the external morphology of 2n pollen was more evolved. In terms of in vitro germination rate and pollen tube length, the induced 2n pollen did not differ significantly from the spontaneous 2n pollen. The survival rate of the floral buds was significantly decreased with increased colchicine concentration and treatment time.

Male sterile 28 encodes an ARGONAUTE family protein essential for male fertility in maize

Male sterility is a common biological phenomenon in plants and is a useful trait for hybrid seed production. Normal tapetum development is essential for viable pollen generation. Although many genes involved in tapetum differentiation and degradation have been isolated in maize, elements that regulate tapetum development during pollen mother cell (PMC) meiosis are less studied. Here, we characterized a classical male-sterile mutant male sterile 28 (ms28) in maize. The ms28 mutant had a regular male meiosis process, while its tapetum cells showed premature vacuolation at the early meiotic prophase stage. Using map-based cloning, we cloned the Ms28 gene and confirmed its role in male fertility in maize together with two allelic mutants. Ms28 encodes the ARGONAUTE (AGO) family protein ZmAGO5c, and its transcripts primarily accumulate in premeiosis anthers, with more intense signals in PMCs. Transcriptomic analysis revealed that genes related to anther development, cell division, and reproductive structure development processes were differentially expressed between the ms28 mutant and its fertile siblings. Moreover, small RNA (sRNA) sequencing revealed that the small interfering RNA (siRNA) and microRNA (miRNA) abundances were obviously changed in ms28 meiotic anthers, which indicated that Ms28 may regulate tapetal cell development through small RNA-mediated epigenetic regulatory pathways. Taken together, our results shed more light on the functional mechanisms of the early development of the tapetum for male fertility in maize.

Reduced fertility caused by meiotic defects and micronuclei formation during microsporogenesis in xBrassicoraphanus

BackgroundHybridization and polyploidization events are important driving forces in plant evolution. Allopolyploids formed between different species can be naturally or artificially created but often suffer from genetic instability and infertility in successive generations. xBrassicoraphanus is an intergeneric allopolyploid obtained from a cross between Brassica rapa and Raphanus sativus, providing a useful resource for genetic and genomic study in hybrid species.ObjectiveThe current study aims to understand the cause of hybrid sterility and pollen abnormality in different lines of synthetic xBrassicoraphanus from the cytogenetic perspective.MethodsAlexander staining was used to assess the pollen viability. Cytogenetic analysis was employed to monitor meiotic chromosome behaviors in pollen mother cells (PMCs). Origins of parental chromosomes in xBrassicoraphanus meiocytes were determined by genome in situ hybridization analysis.ResultsThe xBrassicoraphanus lines BB#4 and BB#6 showed high rates of seed abortion and pollen deformation. Abnormal chromosome behaviors were observed in their PMCs, frequently forming univalents and inter-chromosomal bridges during meiosis. A positive correlation also exists between meiotic defects and the formation of micronuclei, which is conceivably responsible for unbalanced gamete production and pollen sterility.ConclusionThese results suggest that unequal segregation of meiotic chromosomes, due in part to non-homologous interactions, is responsible for micronuclei and unbalanced gamete formation, eventually leading to pollen degeneration and inferior fertility in unstable xBrassicoraphanus lines.

Genome-Wide Identification of lncRNAs Involved in Fertility Transition in the Photo-Thermosensitive Genic Male Sterile Rice Line Wuxiang S.

Long non-coding RNAs (lncRNAs) act as universal regulators of various biological processes, but no genome-wide screening of lncRNAs involved in the fertility transition of the photo-thermosensitive genic male sterile (PTGMS) rice line has been reported. Here, we performed strand-specific RNA sequencing at three developmental stages of a novel PTGMS line Wuxiang S (WXS). A total of 3,948 lncRNAs were identified; 622 of these were detected as differentially expressed lncRNAs (DE-lncRNAs) between male-sterile WXS (WXS-S) and male-fertile WXS (WXS-F). A large proportion of lncRNAs differentially expressed at the stage of pollen mother cells meiosis, suggested that it may be the most critical stage for fertility transition of WXS. Furthermore, functional annotation of the cis- and trans- targets of DE-lncRNAs showed that 150 targets corresponding to 141 DE-lncRNAs were identified as involved in anther and pollen development. Moreover, computational analysis predicted 97 lncRNAs as precursors for 72 miRNAs, and 94 DE-lncRNAs as potential endogenous target mimics (eTMs) for 150 miRNAs. Finally, using the dual luciferase reporter assays, we demonstrated that two lncRNAs act as eTMs to regulate the expression of the SPL and GRF genes by competing for the shared osa-miR156 and osa-miR396, respectively. These genomic characteristics, differential expression, and interaction of lncRNAs with miRNAs and mRNAs contribute to our understanding of the roles of lncRNAs during the fertility transition in PTGMS rice lines.

The Cyto-morphological Studies in Physalis minima Linn.

Cyto-morphological studies in a diploid natural population of Physalis minima Linn were done. It was a diploid natural population recorded behind Jal Mandir on Parasnath hills. Qualitative and quantitative parameters were investigated. Ten morphological characters like height of the plant, number of branches per plant, number of branches from main stem, number of nodes, length of internodes, number of heads per plant, length of leaf, breadth of leaf, length/breadth ratio of leaf andlength of petiole were recorded and statistically analyzed. Cytologically, the population showed, gametic number as n= 12. Most of the pollen mother cells were found to be normal; however, some abnormalities were also recorded. Clumping of chromosomes recorded in both meiotic phases i,e. metaphase-I and anaphase-I. Along with it, at metaphase-I, the anomalies recorded as formation of univalents, multivalents and precocious separation of chromosomes. Anaphase-I showed chromosomal laggards, simple chromosomal bridges and unequal separation of chromosomes as anomalies. The half chiasma per chromosome was 0.87. Pollen sterility was 6.00 percentages.

Meiotic behavior and diploidy in Schinus terebinthifolia (Anacardiaceae)

Abstract The genus Schinus (Anacardiaceae) comprises 30 species found in Peru, Bolivia, Chile, Paraguay, Uruguay, Argentina and Brazil. Schinus terebinthifolia (Brazilian pepper tree) is socioeconomically important in the Brazilian state of Espírito Santo. Dried fruit is used as a pepper-like spice and natural populations are exploited. Studies on the meiotic behavior and pollen stainability from an ecological and evolutionary point of view are essential, since they can promote future sustainable management strategies for this species. The objective was to evaluate meiotic behavior and characteristics of pollen grains of individuals from a natural population of S. terebinthifolia localized in the municipality of Vitória, Espírito Santo state, Brazil. Young anthers were squashed in 1% acetocarmine dye to obtain the pollen mother cells. The pollen stainability with 1% acetocarmine, Lugol’s iodine solution and Sudan IV and pollen shape were used to assess the pollen potential viability, cytochemistry and morphology. All individuals had a gametic number of n = 7, a regular meiotic behavior and so the somatic number is 2n = 2x = 14 chromosomes. Median pollen stainability was 94%. Pollen grains are dispersed as monads, 3 - colporate and subspheroidal. The exine has striate-reticulate ornamentation. Pollen grains are starch positive and lipid negative. This is the first report of the occurrence of diploidy for the species. The high pollen stainability is the result of the occurrence of regular meiosis. The presence of starch as pollen reserve can increase the resistance of pollen grains to hostile environments in this species.

Homoeologous chromosome pairing at metaphase I of meiosis in <i>Hordeum vulgare</i> L. × <i>H. bulbosum</i> L. triploid hybrids (HvHbHb)

Background.One of the ways to use the genetic potential of bulbous barley, which is characterized by a number of valuable traits, is interspecific hybridization. In crosses ofH. vulgare(2x) ×H. bulbosum(2x) andH. vulgare(4x) ×H. bulbosum(4x) with a genome ratio of 1Hv: 1Hb in a hybrid embryo, elimination of bulbous barley chromosomes is observed in many cases, and the intensity of the process and the result of the crossing depend on the genotypes of the parental forms. This limits the possibility of including a significant variety of parental forms in crosses. Сrossing of diploid forms ofH. vulgarewith tetraploid accessions ofH. bulbosum(4x) results in the formation of triploid hybrids (HvHbHb) with stable chromosomal composition in pollen mother cells (PMCs) at metaphase I (MI) of meiosis. These triploid hybrids can serve as a basis for obtaining series of introgressive lines of cultivated barley. One of the tasks of this type of work is to estimate the involvement of various chromosomes and their arms in homoeologous associations. The aim of this work was to study the possibility of homoeologous pairing of chromosomes of parental species at MI of meiosis in triploid hybrids using GISH and FISH with chromosome-specific markers, as well as to register the participation of individual arms of the cultivated barley chromosomes in homoeologous associations with the chromosomes of bulbous barley in triploid hybrids (HvHbHb).Materials and methods.Seven triploid hybrids ofH. vulgare×H.bulbosum(HvHbHb) obtained in four combinations of crosses with the participation of three diploid cultivars of cultivated barley and two tetraploid accession of bulbous barley were used in this study. The features of homoeologous pairing of chromosomes at MI were studied using the method of fluorescentin situhybridization (GISH and FISH) with chromosome-specific markers.ResultsAll the studied hybrid plants are characterized by a stable chromosomal composition in PMCs at the MI stage of meiosis. Meiotic configurations formed by homoeologous chromosomes of the parental species, ranging from 0.87 to 1.40 on average per cell, were identified in all the studied plants. Among them, vbb trivalents prevailed. Analysis of chromosome pairing at MI in triploid hybrids revealed the participation of all chromosome arms ofH. vulgarein homoeologous Hv-Hb associations, except for the short arm of chromosome 1H. In all the studied triploid hybrids, there is a tendency for a higher frequency of involvement of the long arms of chromosomes in the formation of homoeologous associations; this feature is most clearly manifested in case of chromosome 5H.ConclusionsIntergenomic associations with the participation of all arms ofH. vulgarechromosomes, except for the short arm of chromosome 1H, were revealed at MI inH. vulgare×H. bulbosumtriploid hybrids (HvHbHb). Chromosome 5H, as well as any other cultivated barley chromosome, is characterized by a higher involvement of its long arm in homoeologous associations Hv-Hb, as compared to the short arm.

Visualizing Meiotic Chromosome Pairing and Segregation in Interspecific Hybrids of Rice by Genomic in situ Hybridization

Meiotic disturbances in F1 hybrids and their progenies are still major problems in wide hybridization. To investigate the genome affinity reflected in chromosome pairing and segregation, we studied chromosome behaviors during meiosis in two interspecific F1 hybrids [O. minuta×O. australiensis (Om×Oa, BCE genome) and Oa×O. ridleyi (Or, EHJ genome)] by using both traditional staining methods and genomic in situ hybridization (GISH). GISH analysis has been successfully performed on mitotic chromosomes to distinguish different Oryza genomes, but relatively fewer systematic analyses of meiotic chromosomes of interspecific hybrids have been reported. In the hybrids, highly irregular chromosome behaviors through meiosis resulted in producing microspores with unbalanced genome. At diakinesis of these two hybrids, most chromosomes present as univalent, with low frequency as bivalents and occasionally as trivalents. In a pollen mother cell, 2 to 8 bivalents and 0 to 4 trivalents were observed in the hybrid Oa×Or, and 1 to 8 bivalents and 0 to 5 trivalents were observed in the hybrid Om×Oa. GISH results indicated that 51.52% bivalents in Oa×Or and 79.65% bivalents in Om×Oa involved allosyndetic association, which indicates that recombination and introgression should be possible if viable backcrosses can be recovered even from triploid hybrids. In this study, we revealed that the meiotic disturbance due to low affinities between parental genomes is the major reason for the sterility of these two triploid interspecific hybrids. The two hybrids showing vigor in reproductive growth are potential genetic resources in future breeding programs. A better understanding of genomic affinities between these distant Oryza species can facilitate planning an effective breeding program by using wide hybridization, and efficient and routine GISH analysis is helpful to monitor alien introgression in the process.

Morphological characteristics and cytological study of anther abortion of temperature-sensitive nuclear male sterile line 160S in <italic>Brassica napus</italic>

<p indent=0mm>The main purpose of this study was to explore the flower morphologies, anther abortion times and cytological characteristics of the thermo sensitive genic male sterile line 160S in <italic>Brassica napus</italic>. The results will provide a theoretical basis for further study on the molecular regulation mechanism and guide significance for its practical application in two-line hybrid breeding of rape. The 160S were grown at 15℃ and 28℃. The flower morphology, microspore, anther structure and tapetum at different stages of fertile plants (Male Fertile/160S-MF) and sterile plants (Male Sterile/160S-MS) were observed by stereomicroscope with carmine acetate staining, paraffin section, hematoxylin eosin staining and TUNEL staining. The anther was yellow, plump and dehiscent with high pollen viability at 15℃. At 28℃, there was no differences in pistil and potato chips between 160S-MS and 160S-MF flowers, but the petals and the filaments of 160S-MS were smaller and shorter. The stamens of 160S-MS degenerated obviously and its anthers were shriveled in yellowish brown without pollen grains, indicating completely male sterility. But interestingly, the 160S-MF developed normally with higher pollen viability. However, microspore and pollen grains were not observed in 160S-MS due to complete abortion of stamens. Paraffin section results showed that there was no significant difference between 160S-MS and 160S-MF in sporulation and pollen mother cell stages. The morphology and structure of tapetum of 160S-MS appeared some abnormal appearances, such as irregularly cell arrangement, vacuolization, and premature disintegration at meiosis stage. Meanwhile, the development of pollen mother cells were hindered and no tetrads structure were formed leading to the empty pollen sac before the completion of meiosis. TUNEL assay showed that the tapetum of 160-MS began to apoptosis at meiosis stage. In summary, these results showed that 160S belongs to pollen mother cell abortion type male sterile line, whose abortion stage occured during meiosis stage and the tapetum cells degraded prematurely and failed to be transformed to the glandular type, so that the nutrients needed for pollen mother cell development were not provided. The tetrad structures and microspores can not be formed, which leaded to the formation of empty anther sac and resulted in male sterility.

Meiotic studies in &lt;em&gt;Solanum sisymbrifolium&lt;/em&gt; Lamk

Meiotic studies in two populations of Solanum sisymbrifolium were caried out in detail each from Railway track at Gaya railway station and from Lal Darwaja of Munger. Meiosis showed anomalies like multivalent and univalent at metaphase-I and soe pollen other cells showed abnormalities like clumping of chromosomes, precocious separation of chromosomes and translocation chains and rings while at anaphase-I,12:12chromosomes were recorded in most of the pollen mother cells. About 15% of the PMC at this stage were found to be abnormal.

Development of a Yellow-Seeded Stable Allohexaploid Brassica Through Inter-Generic Somatic Hybridization With a High Degree of Fertility and Resistance to Sclerotinia sclerotiorum.

The Brassica coenospeceis have treasure troves of genes that could be beneficial if introgressed into cultivated Brassicas to combat the current conditions of climate change. Introducing genetic variability through plant speciation with polyploidization is well documented, where ploidy augmentation of inter-generic allohexaploids using somatic hybridization has significantly contributed to genetic base broadening. Sinapis alba is a member of the Brassicaceae family that possesses valuable genes, including genes conferring resistance to Sclerotinia sclerotiorum, Alternaria brassicae, pod shattering, heat, and drought stress. This work aimed to synthesize stable allohexaploid (AABBSS) Brassica while incorporating the yellow-seed trait and resistance to S. sclerotiorum stem rot. The two fertile and stable allohexaploids were developed by polyethylene glycol mediated protoplast fusions between Brassica juncea (AABB) and S. alba (SS) and named as JS1 and JS2. These symmetric hybrids (2n = 60) were validated using morphological and molecular cytology techniques and were found to be stable over consecutive generations. The complete chromosome constitution of the three genomes was determined through genomic in situ hybridization of mitotic cells probed with S. alba genomic DNA labeled with fluorescein isothiocyanate. These two allohexaploids showed 24 hybridization signals demonstrating the presence of complete diploid chromosomes from S. alba and 36 chromosomes from B. juncea. The meiotic pollen mother cell showed 30 bivalent sets of all the 60 chromosomes and none of univalent or trivalent observed during meiosis. Moreover, the backcross progeny 1 plant revealed 12 hybridization signals out of a total of 48 chromosome counts. Proper pairing and separation were recorded at the meiotic metaphase and anaphase, which proved the stability of the allohexaploid and their backcross progeny. When screening, the allohexaploid (JS2) of B. juncea and S. alba displayed a high degree of resistance to S. sclerotiorum rot along with a half-yellow and half-brown (mosaic) seed coat color, while the B. juncea and S. alba allohexaplopid1 (JS1) displayed a yellow seed coat color with the same degree of resistance to Sclerotinia rot.

Genetic behavior of induced translocation heterozygote in Artemisia annua L.

From the population raised from gamma irradiated seeds at different doses (100, 200 and 300 Gy), at 100 Gyphenotypically dissimilar plant (from the control plants) was isolated. The plant was shorter and morphologically weaker in contrast to control population. The cytological studies were carried out in a plant showed segmental exchanges between heterologous chromosomes. Chromosomal associations in rings and chains along with bivalents and univalents were observed in pollen mother cells (PMCs) during microsporogenesis. Various anaphasic anomalies were also taken into account including chromatid bridges, laggards and unequal separations etc. Subsequent stages of meiosis were also found to be highly intermittent. Owing to the high frequency of chromosomal aberrations, pollen fertility in control (96.32%) were efficaciously reduced to 38.26% at dose of 100Gy. The present study has been conducted to assess cytological behavior of the induced translocation heterozygote which is first time induced through gamma irradiation in the Artemisia annua L.

Comparative Cytological and Transcriptome Analysis Revealed the Normal Pollen Development Process and Up-Regulation of Fertility-Related Genes in Newly Developed Tetraploid Rice.

Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for its utilization. Here, we reported the development of a new tetraploid rice, Huoduo1 (H1), which has the characteristic of high fertility, from crossing generations of autotetraploid rice. Cytological observations displayed the high fertility of the pollen (95.62%) in H1, a lower percentage of pollen mother cell (PMC) abnormalities, and stable chromosome configurations during the pollen development process compared with its parents. Using RNA-seq analysis, we detected 440 differentially expressed genes (DEGs) in H1 compared with its parents. Of these DEGs, 193 were annotated as pollen fertility-related genes, and 129 (~66.8%) exhibited significant up-regulation in H1 compared with the parents, including three environmentally sensitive genic male sterility genes (TMS9-1, TMS5, and CSA), one meiosis gene (RAD51D), and three tapetal-related genes (MIL2, OsAP25, and OsAP37), which were validated by qRT-PCR in this study. Two genes, TMS9-1 and TMS5, were knocked out using CRISPR/Cas9 technology, and their mutants displayed low fertility and the abnormal development of pollen. Our findings provide evidence for the regulatory mechanisms of fertility in tetraploid rice and indicated that the up-regulation of pollen fertility-related genes may contribute to the high fertility in new tetraploid rice.

Gametophyte and embryonic ontogeny: understanding the reproductive calendar of Cypripedium japonicum Thunb. (Cypripedoideae, Orchidaceae), a lady\u2019s slipper orchid endemic to East Asia

BackgroundThe genus Cypripedium L. is one of the five genera of the subfamily Cypripedioideae, members of which are commonly known as lady’s slipper orchids. Cypripedium japonicum is a perennial herb native to East Asia, specifically China, Japan, and Korea. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List.ResultsWe investigated gametophyte development, including complete embryogenesis, in C. japonicum. The complete reproductive cycle is presented based on our observations. Anther development begins under the soil, and meiosis of pollen mother cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid–late June. The pattern of embryo sac formation is bisporic, and there are six nuclei: three forming the egg apparatus, two polar nuclei, and an antipodal cell in the mature embryo sac. Triple fertilization results in the endosperm nucleus, which degenerates when the proembryo reaches the eight-to-sixteen-cell stage.ConclusionOur overall comparisons of the features of gametophyte and embryo development in C. japonicum suggest that previous reports on the embryology of Cypripedium are not sufficient for characterization of the entire genus. Based on the available information, a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared.

Cell wall Glycine-rich Protein2 is involved in tapetal differentiation and pollen maturation.

The tapetum plays important roles in anther development by providing materials for pollen-wall formation and nutrients for pollen development. Here, we report the characterization of a male-sterile mutant of glycine-rich protein 2 (OsGRP2), which exhibits irregular cell division and dysfunction of the tapetum. GRP is a cellwall structural protein present in the cell walls of diverse plant species, but its function is unclear in pollen development. We found that few GRP genes are expressed in rice and thus focused on one highly expressed gene, OsGRP2. The tapetal cell walls of an OsGRP2 mutant did not thicken at the pollen mothercell stage, as a result, pollen maturation and fertility rate decreased. High OsGRP2 expression was detected in male-floral organs, and OsGRP2 was distributed in the tapetum. OsGRP2 participated in establishment of the cellwall network during early tapetum development. In conclusion, our results indicate that OsGRP2 plays important roles in the differentiation and function of the tapetum.