Abstract: During anther development the male gametophyte develops inside the locule and the tapetal cells provide all nutrients for its development. Magnesium Transporter 5 (MGT5) is a member of the MGT family and has dual functions of Mg export and import. Here, we show that male gametophyte mitosis and intine formation are defective in a mgt5 mutant. The transient expression of GFP-MGT5 revealed that MGT5 is localized in the plasma membrane. These findings suggest that in the male gametophyte MGT5 plays a role in importing Mg from the locule and that Mg is essential for male gametophyte development. The expression of MGT5 in the knockout ABORTED MICROSPORES (AMS) mutant (AMS being an essential regulator of tapetum) is tremendously reduced. Chromatin immunoprecipitation and mobility shift assay experiments demonstrated that AMS can directly bind the promoter of MGT5. An immunoelectron microscopy assay revealed that MGT5-His is localized to the plasma membrane of the tapetum. These findings suggest that AMS directly regulates MGT5 in the tapetum and thus induces export of Mg into the locule. The mgt5 plant exhibits severe male sterility while the expression of MGT5 under the tapetum-specific promoter A9 partly rescued mgt5 fertility. mgt5 fertility was restored under high-Mg conditions. These findings suggest that the mgt5 tapetum still has the ability to export Mg and that a sufficient supply of Mg from the tapetum can improve the importation of Mg in the mgt5 male gametophyte. Therefore, MGT5 plays an important role in Mg transport from the tapetum to the microspore.

Abstract: Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.

Abstract: Pollen, an extremely reduced bicellular or tricellular male reproductive structure of flowering plants, serves as a model for numerous studies covering wide range of developmental and physiological processes. The pollen development represents a fragile and vital phase of plant ontogenesis and pollen was among the first singular plant tissues thoroughly characterized at the transcriptomic level (Honys and Twell [5]). Arabidopsis pollen developmental transcriptome has been published over a decade ago (Honys and Twell, 2004) and transcriptomes of developing pollen of other species have followed (Rice, Deveshwar et al. [2]; Triticeae, Tran et al. [11]; upland cotton, Ma et al. [8]). However, the transcriptomic data describing the development of tobacco pollen, a bicellular model for cell biology studies, have been missing. Here we provide the transcriptomic data covering three stages (Tupý et al., 1983) of wild type tobacco (Nicotiana tabacum, cv. Samsun) pollen development: uninucleate microspores (UNM, stage 1), early bicellular pollen (eBCP, stage 3) and late bicellular pollen (lBCP, stage 5) as a supplement to the mature pollen (MP), 4 h-pollen tube (PT4), 24 h-pollen tubes (PT24), leaf (LF) and root (RT) transcriptomic data presented in our previous studies (Hafidh et al., 2012a; Hafidh et al., 2012b). We characterized these transcriptomes to refine the knowledge base of male gametophyte-enriched genes as well as genes expressed preferentially at the individual stages of pollen development. Alongside updating the list of tissue-specific genes, we have investigated differentially expressed genes with respect to early expressed genes. Pollen tube growth and competition of pollen tubes in female pistil can be viewed as a race of the fittest. Accordingly, there is an apparent evolutionary trend among higher plants to store significant material reserves and nutrients during pollen maturation. This supply ensures that after pollen germination, the pollen tube utilizes its resource predominantly for its rapid elongation in the female pistil. Previous transcriptomic data from Arabidopsis showed massive expression of genes encoding proteins forming both ribosomal subunits that were accumulated in developing pollen, whereas their expression was not detectable in growing pollen tubes (Honys and Twell, 2004). We observed a similar phenomenon in less advanced bicellular tobacco pollen. Here, we describe in detail how we obtained and analyzed validated microarray dataset deposited in Gene Expression Omnibus (GSE62349).

Abstract: Ovary pre-conditioned medium and ovary co-culture increased the efficiency of green doubled haploid plant production in bread wheat anther culture. The positive effect of this medium led to a 6- and 11-fold increase in the numbers of embryos and green plants, respectively, having a greater effect on a medium-low responding cultivar. Ovary genotype and developmental stage significantly affected microspore embryogenesis. By the use of Caramba ovaries it was possible to reach a 2-fold increase in the number of embryos and green plants, and to decrease the rate of albinism. Mature ovaries from flowers containing microspores at a late binucleate stage raised the number of embryos and green plants by 25-46% as compared to immature ovaries (excised from flowers with microspores at a mid-late uninucleate stage). The highest numbers of embryos and green plants were produced when using mature Caramba ovaries. Ovaries from Galeon, Tigre, and Kilopondio cultivars successfully induced microspore embryogenesis at the same rate as Caramba ovaries. Moreover, Tigre ovaries raised the percentage of spontaneous chromosome doubling up to 71%. Attempts were made to identify molecular mechanisms associated to the inductive effect of the ovaries on microspore embryogenesis. The genes TAA1b, FLA26, and WALI6 associated to wheat microspore embryogenesis, the CGL1 gene involved in glycan biosynthesis or degradation, and the FER gene involved in the ovary signaling process were expressed and/or induced at different rates during ovary culture. The expression pattern of FLA26 and FER could be related to the differences between genotypes and developmental stages in the inductive effect of the ovary. Our results open opportunities for new approaches to increase bread wheat doubled haploid production by anther culture, and to identify the functional components of the ovary inductive effect on microspore embryogenesis.

Abstract: Microspores are specialized generative cells with haploid genome that demonstrate the amenability toward embryogenesis under certain conditions. The induced microspore culture technique is largely exploited by the breeding programs of wheat and other crops due to its high efficiency for generation of the large number of haploid plants in the relatively short period of time. The ability to produce mature double haploid plant from a single cell has also attracted attention of the plant biotechnologists in the past few years. More importantly, the possibility to deliver proteins for improvement of embryogenesis and the genome modification purposes holds great potential for transgene-free wheat biotechnology. In the present study, we examined the ability of cationic and amphipathic cell penetrating peptides (CPPs) to convey a covalently-linked mCherry protein inside the viable microspores. We demonstrate that the affinity of CPPs to the microspore cells dependents on their charge with the highest efficiency of CPP-mCherry binding to the cells achieved by cationic CPPs (penetratin and R9). Additionally, due to overall negative charge of the microspore cell wall, the successful uptake of the protein cargo by live microspore cells is attained by utilization of a reversible disulfide bond between the R9 CPP and mCherry protein. Overall, the approach proposed herein can be applied by the other biotechnology groups for the fast and efficient screening of the different CPP candidates for their ability to deliver proteins inside the viable plant cells.

Abstract: Microspore embryogenesis is a method of achieving complete homozygosity from plants. It is particularly useful for woody species, like Citrus, characterized by long juvenility, a high degree of heterozygosity and often self-incompatibility. Anther culture is currently the method of choice for microspore embryogenesis in many crops. However, isolated microspore culture is a better way to investigate the processes at the cellular, physiological, biochemical, and molecular levels as it avoids the influence of somatic anther tissue. To exploit the potential of this technique, it is important to separate the key factors affecting the process and, among them, culture medium composition and particularly the plant growth regulators and their concentration, as they can greatly enhance regeneration efficiency. To our knowledge, the ability of meta-Topolin, a naturally occurring aromatic cytokinin, to induce gametic embryogenesis in isolated microspores of Citrus has never been investigated. In this study, the effect of two concentrations of meta-Topolin instead of benzyladenine or zeatin in the culture medium was investigated in isolated microspore culture of two genotypes of Citrus. After 11 months of isolated microspore culture, for both genotypes and for all the four tested media, the microspore reprogramming and their sporophytic development was observed by the presence of multinucleated calli and microspore-derived embryos at different stages. Microsatellite analysis of parental and embryo samples was performed to determine the embryo alleles constitution of early embryos produced in all tested media, confirming their origin from microspores. To our knowledge, this is the first successful report of Citrus microspore embryogenesis with isolated microspore culture in Citrus, and in particular in Citrus clementina Hort. ex Tan, cvs. 'Monreal Rosso' and 'Nules.'

Abstract: Using light microscope (LM) and scanning electron microscope (SEM), the megaspores and microspores of 77 samples representing ca. 70 species of Selaginella from China are observed. Combing previous studies, the spore morphology of nearly all documented Selaginella species from China were reviewed. Based on the morphological characteristics in megaspores and/or microspores, we divided the spores of Chinese species into 15 types and three types are further divided into various subtypes. Each type and subtype are described in detail and a key to the types and subtypes of spores is given. For the first time, the systematic significance of microspores of Selaginella are discussed, and the results indicate that microspores of Selaginella are significant in the systematics of Selaginella . Some important morphological characteristics in spores (e.g., color, micro-sculpture, size, etc.), often been neglected in previous studies, are introduced. Some spore-morphological synapomorphies of the clades and subclades, identified by recent molecular work (Zhou et al. 2015a), are well established. Using the spore morphology, the delimitation of some taxonomically difficult species in Selaginella is assessed.

Abstract: Doubled haploid technology is an important tool in plant breeding and research, but routine application requires the establishment of efficient protocols. Micro- spore culture is an attractive approach although its effi- ciency is strongly dependent on the genotype. In this study we evaluated the effects of the three ethylene inhibitors aminooxyacetic acid, 2,5-norbornadiene, and silver thio- sulphate (STS) on embryogenesis, regeneration rate and green plant rate in triticale microspore culture. Our results show that STS at 20 lM in particular had a positive effect on the embryogenesis rate, suggesting that for many genotypes ethylene accumulates in the culture vessel above a critical threshold where it becomes inhibitory for embryogenesis. In addition, STS also appears to positively influence the green plant rate. Taken together, our results show that the addition of ethylene inhibitors at defined concentrations can counteract the negative effect of ethy- lene, increase the embryogenesis rate and potentially also the green plant rate in microspore culture.

Abstract: In this study, we reported that a F-box protein, OsADF, as one of the direct targets of TDR , plays a critical role in rice tapetum cell development and pollen formation. The tapetum, the innermost sporophytic tissue of anther, plays an important supportive role in male reproduction in flowering plants. After meiosis, tapetal cells undergo programmed cell death (PCD) and provide nutrients for pollen development. Previously we showed that tapetum degeneration retardation (TDR), a basic helix-loop-helix transcription factor, can trigger tapetal PCD and control pollen wall development during anther development. However, the comprehensive regulatory network of TDR remains to be investigated. In this study, we cloned and characterized a panicle-specific expression F-box protein, anther development F-box (OsADF). By qRT-PCR and RNA in situ hybridization, we further confirmed that OsADF expressed specially in tapetal cells from stage 9 to stage 12 during anther development. In consistent with this specific expression pattern, the RNAi transgenic lines of OsADF exhibited abnormal tapetal degeneration and aborted microspores development, which eventually grew pollens with reduced fertility. Furthermore, we demonstrated that the TDR, a key regulator in controlling rice anther development, could regulate directly the expression of OsADF by binding to E-box motifs of its promoter. Therefore, this work highlighted the possible regulatory role of TDR, which regulates tapetal cell development and pollen formation via triggering the possible ADF-mediated proteolysis pathway.

Abstract: The induction of microspore embryogenesis produces dramatic changes in different aspects of the cell physiology and structure. Changes at the cell wall level are among the most intriguing and poorly understood. In this work, we used high pressure freezing and freeze substitution, immunolocalization, confocal and electron microscopy to analyze the structure and composition of the first cell walls formed during conventional Brassica napus microspore embryogenesis, and in cultures treated to alter the intracellular Ca2+ levels. Our results revealed that one of the first signs of embryogenic commitment is the formation of a callose-rich, cellulose-deficient layer beneath the intine (the subintinal layer), and of irregular, incomplete cell walls. In these events, Ca2+ may have a role. We propose that abnormal cell walls are due to a massive callose synthesis and deposition of excreted cytoplasmic material, and the parallel inhibition of cellulose synthesis. These features were absent in pollen-like structures and in microspore-derived embryos, few days after the end of the heat shock, where abnormal cell walls were no longer produced. Together, our results provide an explanation to a series of relevant aspects of microspore embryogenesis including the role of Ca2+ and the occurrence of abnormal cell walls. In addition, our discovery may be the explanation to why nuclear fusions take place during microspore embryogenesis.

Abstract: The male germline of flowering plants constitutes a specialized lineage of diminutive cells initiated by an asymmetric division of the initial microspore cell that sequesters the generative cell from the pollen vegetative cell. The generative cell subsequently divides to form the two male gametes (non-motile sperm cells) that fuse with the two female gametophyte target cells (egg and central cells) to form the zygote and endosperm. Although these male gametes can be as little as 1/800th of the volume of their female counterpart, they encode a highly distinctive and rich transcriptome, translate proteins, and display a novel suite of gamete-distinctive control elements that create a unique chromatin environment in the male lineage. Sperm-expressed transcripts also include a high proportion of transposable element-related sequences that may be targets of non-coding RNA including miRNA and silencing elements from peripheral cells. The number of sperm-encoded transcripts is somewhat fewer than the number present in the egg cell, but are remarkably distinct compared to other cell types according to principal component and other analyses. The molecular role of the male germ lineage cells is just beginning to be understood and appears more complex than originally anticipated.

Abstract: The change in developmental fate of microspores reprogrammed towards embryogenesis is a complex but fascinating experimental system where microspores undergo dramatic changes derived from the developmental switch. After 40 years of study of the ultrastructural changes undergone by the induced microspores, many questions are still open. In this work, we analyzed the architecture of DNA-containing organelles such as plastids and mitochondria in samples of B. napus isolated microspore cultures covering the different stages before, during and after the developmental switch. Mitochondria presented a conventional oval or sausage-like morphology for all cell types studied, similar to that found in vivo in other cell types from vegetative parts. Similarly, plastids of microspores before induction and of non-induced cells showed conventional architectures. However, approximately 40% of the plastids of embryogenic microspores presented atypical features such as curved profiles, protrusions, and internal compartments filled with cytoplasm. Three-dimensional reconstructions confirmed that these plastids actually engulf cytoplasm regions, isolating them from the rest of the cell. Acid phosphatase activity was found in them, confirming the lytic activity of these organelles. In addition, digested plastid-like structures were found excreted to the apoplast. All these phenomena seemed transient, since microspore-derived embryos showed conventional plastids. Together, these results strongly suggested that under special circumstances, such as those of the androgenic switch, plastids of embryogenic microspores behave as autophagic plastids (plastolysomes), engulfing cytoplasm for digestion, and then are excreted out of the cytoplasm as part of a cleaning program necessary for microspores to become embryos.

Abstract: Albinism is a major limitation in the production of doubled haploid plants in cereals. As this trait is partly genetically controlled, the culture method has to be adapted to the plant genotype to reduce albinism. We have improved green plant recovery in the Northern barley ( Hordeum vulgare L.) spring cultivar Mitja from 6 to 42% by altering the culture method and composition of the culture media. In a new three-step protocol, the addition of casein hydrolysate to an inter - mediate regeneration medium gave the high - est number of green plants. The shoot and root growth medium used for germinated embryos was superior to the standard media reported in the literature for root formation and shoot growth. o ur modified method has merit for test - ing with other recalcitrant genotypes in anther and isolated microspore cultures.

Abstract: Anther development is very precise and complicated procedure. In this study, the gene LoMYB80 was isolated from lily (Lilium Oriental Hybrids cv. cultivar ‘Siberia’) anther. Multiple alignment and phylogenetic tree analysis showed that LoMYB80 belongs to the R2R3 MYB family. Furthermore, the protein of LoMYB80 shares a 44 amino acid sequence immediately adjacent to the R3 domain with other MYB80s. Subcellular localization showed that LoMYB80 was predominantly localized in the nucleus. The transcriptional activation experiment revealed that LoMYB80 belongs to the transcription activation factors and its transactivation activity depends on the last 70 amino acids located at the end of the C-terminal region. Real-time quantitative PCR analysis showed that LoMYB80 gene expression was significantly higher in anther than in other organs; the highest level was in anthers of 10-cm flower buds and then was in anthers of 4-cm buds. The relative expression in microspores/pollen of 10-cm buds was much higher than the whole anther in the same stage and also much higher than the microspore/pollen of 8- and 9-cm buds. The gene was mainly expressed in young anthers at the development stages from 6 to 10 by analyzing transgenic Arabidopsis lines which were transformed with LoMYB80 promoter::GUS reporter gene constructs. Complementation experiments revealed that transgenic ms188 mutant lines could produce normal pollen grains and display partial fertility. These results indicated that LoMYB80 was a regulator of lily anther development and pollen formation.

Abstract: Haploid plants provide an excellent example of cellular totipotency as they develop from either the male or female gametes without fertilization. Haploids can be induced in vivo and in vitro. Androgenesis is the development of haploids from male gametes. The two main techniques that are employed to generate androgenic plants in vitro are (a) anther culture and (b) microspore culture. The application of anther culture is widespread. Different developmental pathways of microspore embryogenesis have been reported in different plant species and in response to different stress treatments and its microspore developmental stage. Genotype or physiology of the donor plant, microspore developmental stage, stress pretreatment, culture medium and culture conditions affect the androgenesis process. Each crop has individual optimal pretreatment regimes and in vitro culture requirements. In vivo haploid induction techniques involve parthenogenesis and wide hybridization followed by chromosome elimination. In maize (Zea mays L.), haploids are commonly produced by in vivo haploid induction system and the method has been commercially exploited by both public and private sector organizations. Wide hybridization method is limited to potato and cereals. Wheat, barley and potato are excellent examples of wide hybridization followed by chromosome elimination system of haploid induction. The regenerated plants particularly in androgenesis need ploidy analysis to distinguish haploids from non-haploid plants. Regardless of the mode of development, doubling of haploid plants either spontaneously or by chemical means leads to a homozygous doubled haploid (DH) individual with two identical copies of each chromosome. Doubled haploids have been used in breeding programmes to produce homozygous genotypes in a number of important species and the number is increasing. DH populations are ideal for genetic mapping and allow the development of high-density marker maps that can then be exploited in quantitative trait locus identification. Genetic mapping using DHs had an impact in locating gene control traits for yield, quality, agronomy, abiotic and biotic stress. DHs now feature in cultivar production in a number of crops, and breeding time is considerably reduced. Hence, worldwide use of DH technology as an accelerated approach to crop improvement has become routine by many breeding companies and laboratories leading to the development of almost 300 new varieties.

Abstract: Nsa cytoplasmic male sterility (CMS) is a novel Brassica napus male sterility system derived from Sinapis arvensis cytoplasm. Nsa CMS results in defective pollen production due to S. arvensis mitochondrial gene failure/incompatibility in the anthers, requiring nuclear genes to restore fertility. From ultramicroscopic observation of anther sections, we concluded that the induction of sterility begins at the pollen (microspore) mother cell stage. Most pollen mother cells do not undergo the first meiotic division and dissociate before the tetrad stage. At the tetrad stage, abortion was observed for all uninucleate pollen. Dysfunction of mitchondrial gene(s) leads to cell vacuolization in the anther tapetum and middle layer cells. Early programmed cell death (PCD) of the tapetum and anther middle layer cells is the main reason for pollen mother cell abortion. Due to this early pollen abortion, the sterility of Nsa CMS is stable and complete. Southern blotting of DNA from the Nsa male-sterile line, its maintainer and restorer lines, as well as the two parental lines (B. napus cv. Zhongshuang 4 and S. arvensis var. Yeyou 18) involved in the somatic hybridization, suggested that the cytoplasm of the Nsa CMS line was from S. arvensis and that of the restorer line was a rearrangement of both parental lines. Nsa CMS shows great potential for hybrid seed production in rapeseed. Our results provide clues to identify novel male-sterility (S) and restorer (R) genes as well as elucidate the mechanism underlying interactions between the S and R genes.

Abstract: Understanding how herbicides affect plant reproduction and growth is critical to develop herbicide toxicity model and refine herbicide risk assessment. Although our knowledge of herbicides toxicity mechanisms at the physiological and molecular level in plant vegetative phase has increased substantially in the last decades, few studies have addressed the herbicide toxicity problematic on plant reproduction. Here, we determined the long-term (4–8 weeks) effect of a chiral herbicide, imazethapyr (IM), which has been increasingly used in plant crops, on floral organ development and reproduction in the model plant Arabidopsis thaliana. More specifically, we followed the effect of two IM enantiomers (R- and S-IM) on floral organ structure, seed production, pollen viability and the transcription of key genes involved in anther and pollen development. The results showed that IM strongly inhibited the transcripts of genes regulating A. thaliana tapetum development (DYT1: DYSFUNCTIONAL TAPETUM 1), tapetal differentiation and function (TDF1: TAPETAL DEVELOPMENT AND FUNCTION1), and pollen wall formation and developments (AMS: ABORTED MICROSPORES, MYB103: MYB DOMAIN PROTEIN 103, MS1: MALE STERILITY 1, MS2: MALE STERILITY 2). Since DYT1 positively regulates 33 genes involved in cell-wall modification (such as, TDF1, AMS, MYB103, MS1, MS2) that can catalyze the breakdown of polysaccharides to facilitate anther dehiscence, the consistent decrease in the transcription of these genes after IM exposure should hamper anther opening as observed under scanning electron microscopy. The toxicity of IM on anther opening further lead to a decrease in pollen production and pollen viability. Furthermore, long-term IM exposure increased the number of apurinic/apyrimidinic sites (AP sites) in the DNA of A. thaliana and also altered the DNA of A. thaliana offspring grown in IM-free soils. Toxicity of IM on floral organs development and reproduction was generally higher in the presence of the R-IM enantiomer than of the S-IM enantiomer. This study unraveled several IM toxicity targets and mechanisms at the molecular and structural level linked to the toxicity of IM trace concentrations on A. thaliana reproduction.

Abstract: This paper presents a comparative description of the starch distribution in the anthers, microspores and pollen grains of Aechmea recurvata, Dyckia racinae and Tillandsia aeranthos. Flowers at different stages of development were processed according to plant microtechniques for observation by light microscope. Ten stages of embryological development were used as references for the comparative analysis of starch distribution and dynamics. The structural data showed a greater starch accumulation in the parietal layers and connective of D. racinae. It was observed that in the species studied, starch began to accumulate in microspore mother cell stage. The pollen grains in D. racinae and in T. aeranthos present two amylogenesis-amylolysis cycles, while A. recurvata presents only one. One amylogenesis-amylolysis cycle occurs in the parietal layers and/or connective tissue in all three species. The pollen grains in the three species are dispersed without starch and are characterized as the starchless type. Starch dynamics presents a close relation to the development of sporangia, microspores and pollen grains. It is believed that differences in the starch distribution and accumulation are related to the abiotic factors where the species are found.

Abstract: Doubled haploids (DHs) production through androgenesis is a biotechnological method for genetic improvement of crops. Biotechnological DH line production offers advantages to plant breeders, including the possibility to obtain homozygous lines within a year in contrast to common inbreeding methods, which may take 6–12 years. The greatest success in androgenesis has been achieved in some varieties of rapeseed ( Brassica napus L.). However, the efficiency of androgenesis in other Brassica species is still poor. Induction of microspore embryogenesis is usually induced by many factors such as conditions of donor plant growth, genotype, microspore developmental stage, culture medium composition, and culture conditions. The reprogramming of microspores from the gametophytic to the sporophytic habit of development depends on various stress factors. Certain pretreatments of microspores, such as high temperature and colchicine, can favor androgenesis in Brassica species. Plant regeneration from microspores can be improved by proper application of different growth regulators (ethylene, abscisic acid, and indole acetic acid). Optimal combinations of these factors are mandatory for efficient androgenesis. In this review, we summarize the experience of our colleagues in DH-technology in the Brassica genus. Attention is focused on some factors influencing the development of doubled haploid plants and their impact on enhancing the efficiency of androgenesis in Brassica species.

Abstract: Though cytoplasmic male sterility (CMS) in peppers is associated with the orf507 gene, definitive and direct evidence that it directly causes male sterility is still lacking. In this study, differences in histochemical localization of anther cytochrome c oxidase between the pepper CMS line and maintainer line were observed mainly in the tapetal cells and tapetal membrane. Inducible and specific expression of the orf507 gene in the pepper maintainer line found that transformants were morphologically similar to untransformed and transformed control plants, but had shrunken anthers that showed little dehiscence and fewer pollen grains with lower germination rate and higher naturally damaged rate. These characters were different from those of CMS line which does not produce any pollen grains. Meanwhile a pollination test using transformants as the male parent set few fruit and there were few seeds in the limited number of fruits. At the tetrad stage, ablation of the tapetal cell induced by premature programmed cell death occurred in the transformants and the microspores were distorted and degraded at the mononuclear stage. Stable transmission of induced semi-male sterility was confirmed by a test cross. In addition, expression of orf507 in the maintainer lines seemed to inhibit expression of atp6-2 to a certain extent, and lead to the increase of the activity of cytochrome c oxidase and the ATP hydrolysis of the mitochondrial F1Fo-ATP synthase. These results introduce the premature programmed cell death caused by orf507 gene in tapetal cells and semi-male sterility, but not complete male sterility.

Abstract: Globular 32 and 64 celled embryoids were obtained from uninucleate apple microspores (cultivar Jonathan) after 5 weeks of culture on a modified Murashige and Skoog (1962) medium. A similar induction of microspore development was not observed in younger or older stages of anther development. In such anthers only callus was formed from diploid tissues.

Abstract: Photoperiod and/or temperature-sensitive male sterility is an economically effective pollination control system for hybrid crops production. This study reports some characteristics of a temperature-sensitive genic male sterile (TGMS) in Brassica napus line, SP2S, which is a spontaneous mutation found in 2007. The fertility alteration in response to temperature was investigated under controlled environment conditions. The pollen abortion was observed under light and electron microscopes to investigate structural and cellular changes associated with male sterility. The results showed that the fertility of SP2S line was greatly influenced by temperature change 12–14 days prior to flowering while photoperiod had no obvious effect on it. The SP2S line became male sterile when daily maximum temperature was above 20 °C and nearly fertile when lower than 15 °C. The size of petal and stamen reduced while the pistil and nectary was normal with good seed set after open-pollination. Microscopic observation in this study revealed the extremely vacuolated and enlarged tapetal cells of PMC during meiosis, which was associated with pollen abortion. The microspores were bonded together and hardly released from tetrads due to the persistence of callose layer covering them. The cytoplasm of degenerated microspores was accompanied by early degraded tapetum. The fertility of SP2S was controlled by at least two recessive nuclear genes and all cultivars could restore the fertility of SP2S. Commercially valuable traits such as seed yield, seed quality, sclerotinia-resistance, lodging, and other agronomical traits of four F1 hybrids of SP2S were as good as their counterparts from near isogenic line, SP2F. The recessive TGMS genes showed no adverse effect on the performance of F1 hybrids. Thus, SP2S line is a promising TGMS for hybrid production in rapeseed and canola. This characterization study helped understanding the process of male sterility and producing super hybrids through this two-line pollination control system.

Abstract: The Cichorium intybus flower development in fertile, cytoplasmic male sterility (CMS 524) and various phenotypes carrying the 524 male sterile cytoplasm was investigated macroscopically and by light microscopy. The development was similar in fertile and in male sterile florets up to meiosis, and then it was affected in anther wall structure and pollen grain development in male sterile floret. In the male sterile plants, the tapetum intrusion after meiosis was less remarkable, the microspores started to abort at vacuolate stage, the connective tissue collapsed, and endothecium failed to expand normally and did not undergo cell wall lignification, which prevented anther opening since the septum and stomium were not disrupted. Crosses undertaken in order to introduce the CMS 524 into two different nuclear backgrounds gave rise to morphologically diversified progenies due to different nuclear-mitochondrial interactions. Macroscopic and cytological investigations showed that pollen-donor plants belonging to Jupiter population had potential capacity to restore fertility while the CC line could be considered as a sterility maintainer.

Abstract: In flowering plants, the male and female gametogenesis is a crucial step of sexual reproduction. Although many genes have been identified as being involved in the gametogenesis process, the genetic mechanisms underlying gametogenesis remains poorly understood. We reported here characterization of the gene, ABORTED GAMETOPHYTE 1 (AOG1) that is newly identified as essential for gametogenesis in Arabidopsis thaliana. AOG1 is expressed predominantly in reproductive tissues including the developing pollen grains and ovules. The AOG1 protein shares no significant amino acid sequence similarity with other documented proteins and is located mainly in nuclei of the cells. Mutation in AOG1 caused degeneration of pollen at the uninucleate microspore stage and severe defect in embryo sacs, leading to a significant reduction in male and female fertility. Furthermore, the molecular analyses showed that the aog1 mutant significantly affected the expression of several genes, which are required for gametogenesis. Our results suggest that AOG1 plays important roles in gametogenesis at the stage prior to pollen mitosis I (PMI) in Arabidopsis, possibly through collaboration with other genes.

Abstract: Pollen developing process was studied to identify the stages of pollen degeneration and to elucidate the factors controlling low pollen fertility in three functional cytoplasmic male-sterile (CMS) lines of eggplant ‘Uttara’. The CMS lines of eggplant were developed by repeated backcrossing using the cytoplasms of wild Solanum species S. kurzii Brace & Prain, S. violaceum Ort., and S. virginianum L.. Anthers were squashed in 1% aceto-carmine to assess pollen staining ability and pollen degeneration at different stages of development. Unicellular microspores were released from tetrads after normal meiotic division. Pollen degeneration occurred at different stages of pollen development in CMS lines such as at unicellular microspore (29.3–36.3%), early bicellular pollen (5.5–12.2%), and late bicellular pollen (9.3–10.2%) stages. On the other hand, in eggplant, only 3.8% pollen was degenerated at unicellular microspore stage and there was negligible pollen degeneration at other stages. Among the stained pollen, abnormally stained (partly and faintly) pollen were found significantly higher in the CMS lines as compared to eggplant. Well stained pollen was varied from 23.2–31.9% in the CMS lines which was significantly lower than that of eggplant. Number of pollens per anther of CMS lines did not vary significantly from eggplant, except the CMS line with S. virginianum cytoplasm. In vitro pollen germination rate in the CMS lines was found to be significantly lower than that of eggplant. Starch accumulation and hydrolysis during pollen maturation were found incomplete in the CMS lines. Degeneration of pollen in different stages, abnormally stained pollen, incomplete starch accumulation and hydrolysis were most likely causes for low pollen fertility in these three CMS lines of eggplant.

Abstract: SUMMARYMicrospore embryogenesis in plants is a process by which microspores can switch their developmental pathway from gametophytic to embryogenic via specific inductive stress treatments, which results in the formation of haploid embryos. However, only a small portion of the cultured cells can undergo the entire process to produce embryos, and most die at different developmental stages during culture. The objective of this study was to improve the induction of embryogenesis in an isolated microspore culture (IMC) of white cabbage (Brassica oleracea L. var. capitata) by focussing on the regulation of dead microspores. The results indicated that microspores died rapidly after 24 h at a heat shock temperature of 32.5°C, and that 80–90% were dead after 3 d in culture. Cell purification performed after 3 d in culture, eliminated most of the dead microspores and had a beneficial effect on embryo yield. Similarly, ascorbic acid (AsA) had a positive effect on the number of embryos produced by decreasing the mor...

Abstract: The plant species Camellia oleifera is an important producer of edible oil in China. However, it suffers from a low fruit-setting rate. This study used high resolution scanning electron microscopy of semi-thin tissue sections to investigate anther development and pollen formation, and to determine whether problems in forming functional pollen cause low seed setting rates. During anther development, cell sizes within the epidermis and end othecium gradually increase, and at the beginning of the microsporocyte phase, the cells become highly vacuolated. The cell walls of the inner wall cells thicken in a ribbon pattern during the later part of the two-cell pollen stage. Middle layer cells become flattened during the later part of the secondary sporogenous cell stage. A tapetum also forms at this stage; the secondary sporogenous cells begin to degrade at the early microspore stage and completely disappear by the time the pollen is mature, thus forming a glandular tapetum. In microsporocyte, cytokinesis occurs simultaneously with microsporocyte meiosis, and a tetrahedral arrangement is present in tetrads. Reproductive cells begin to form at the later stage of microsporogenesis; the nuclei of the reproductive cells are enlarged as the reproductive cells begin to move away from the pollen wall. Two-cell pollen is first formed after reproductive cells have left the pollen wall, and the number of inclusions in the pollen subsequently increases. The pollen matures for 5-8 days before flowering occurs. No pollen abortion phenomena were observed, suggesting that anther development in C. oleifera is normal and that functional pollen are formed.

Abstract: Homozygous lines are very interesting for genetic studies and in plant breeding. These can be produced in a single step via haploidy. Production of haploids in loquat (Eriobotrya japonica (Thunb.) Lindl.) would be of considerable value. In this study, androgenesis by anther culture was used to regenerate loquat plants. Different variables related to the induction of embryogenesis were tested, including the temperature pretreatment of flower buds, various levels of growth regulators in the culture medium, and genotypic effects. Eight cultivars of loquat from different geographic origins were used. The first step, the association between floral bud size and the corresponding microspore/pollen developmental stages was established for the different cultivars. Bud sizes of 6.5–7.0 mm, corresponded to the uninucleate microspore stage and provided the highest callogenesis levels. Pre-treatment of flower buds at 4 °C for had a negative effect on anther response and only the 4-day pre-treatment resulted in callus formation at all. The highest percentage of morphogenetic calli was obtained on medium supplemented with 4.56 μM zeatin (Z) and 5.36 μM 1_naphthalene acetic acid (NAA) in cvs. ‘Changhong-3’ (27 %), ‘Jiefanghong’ (30 %) and ‘Moggi Wase’ (36 %). Following a transfer of morphogenetic calli to the embryo induction medium, six embryos were obtained from cv. ‘Jiefanghong’; and one of them developed into a plantlet. Flow cytometry and chromosome counts revealed that the plantlet was triploid. The single triploid plant obtained was unexpected and its origin is obscure. One possible explanation is that it arose from fusion of sperm nucleus and two vegetative nuclei.