Abstract: We provide evidence on the localization, synthesis, transport, and effects of auxin on the processes occurring late in Arabidopsis thaliana stamen development: anther dehiscence, pollen maturation, and preanthesis filament elongation. Expression of auxin-sensitive reporter constructs suggests that auxin effects begin in anthers between the end of meiosis and the bilocular stage in the somatic tissues involved in the first step of dehiscence as well as in the microspores and in the junction region between anther and filament. In situ hybridizations of the auxin biosynthetic genes YUC2 and YUC6 suggest that auxin is synthesized in anthers. In agreement with the timing of auxin effects, the TIR1, AFB1, AFB2, and AFB3 auxin receptor-encoding genes are transcribed in anthers only during late stages of development starting at the end of meiosis. We found that in tir1 afb triple and quadruple mutants, anther dehiscence and pollen maturation occur earlier than in the wild type, causing the release of mature pollen grains before the completion of filament elongation. We also assessed the contribution of auxin transport to late stamen developmental processes. Our results suggest that auxin synthesized in anthers plays a major role in coordinating anther dehiscence and pollen maturation, while auxin transport contributes to the independent regulation of preanthesis filament elongation.

Abstract: During microsporogenesis, the microsporocyte (or microspore) plasma membrane plays multiple roles in pollen wall development, including callose secretion, primexine deposition, and exine pattern determination. However, plasma membrane proteins that participate in these processes are still not well known. Here, we report that a new gene, RUPTURED POLLEN GRAIN1 (RPG1), encodes a plasma membrane protein and is required for exine pattern formation of microspores in Arabidopsis (Arabidopsis thaliana). The rpg1 mutant exhibits severely reduced male fertility with an otherwise normal phenotype, which is largely due to the postmeiotic abortion of microspores. Scanning electron microscopy examination showed that exine pattern formation in the mutant is impaired, as sporopollenin is randomly deposited on the pollen surface. Transmission electron microscopy examination further revealed that the primexine formation of mutant microspores is aberrant at the tetrad stage, which leads to defective sporopollenin deposition on microspores and the locule wall. In addition, microspore rupture and cytoplasmic leakage were evident in the rpg1 mutant, which indicates impaired cell integrity of the mutant microspores. RPG1 encodes an MtN3/saliva family protein that is integral to the plasma membrane. In situ hybridization analysis revealed that RPG1 is strongly expressed in microsporocyte (or microspores) and tapetum during male meiosis. The possible role of RPG1 in microsporogenesis is discussed.

Abstract: *† These authors contributed equally to this work. Summary Members of the glucan synthase-like (GSL) family are believed to be involved in synthesis of the cell-wall component callose in specialized locations throughout the plant. We identified two members of the Arabidopsis GSL gene family, GSL8 and GSL10, that are independently required for male gametophyte development and plant growth. Analysis of gsl8 and gsl10 mutant pollen during development revealed specific malfunctions associated with asymmetric microspore division. GSL8 and GSL10 are not essential for normal microspore growth and polarity, but play a role in entry of microspores into mitosis. Impaired function of GSL10 also leads to perturbation of microspore division symmetry, irregular callose deposition and failure of generative-cell engulfment by the cytoplasm of the vegetative cell. Silencing of GSL8 or GSL10 in transgenic lines expressing gene-specific dsRNAi constructs resulted in a dwarfed growth habit, thereby revealing additional and independent wild-type gene functions for normal plant growth.

Abstract: The male gametophyte and tapetum play different roles during anther development although they are differentiated from the same cell lineage, the L2 layer. Until now, it has not been possible to delineate their transcriptomes due to technical difficulties in separating the two cell types. In the present study, we characterized the separated transcriptomes of the rice microspore/pollen and tapetum using laser microdissection (LM)-mediated microarray. Spatiotemporal expression patterns of 28,141 anther-expressed genes were classified into 20 clusters, which contained 3,468 (12.3%) anther-enriched genes. In some clusters, synchronous gene expression in the microspore and tapetum at the same developmental stage was observed as a novel characteristic of the anther transcriptome. Noteworthy expression patterns are discussed in connection with gene ontology (GO) categories and gene annotations, which are related to important biological events in anther development, such as pollen maturation, pollen germination, pollen tube elongation and pollen wall formation.

Abstract: In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. Previously, we cloned a pollen specific gene, zm401, from a cDNA library generated from the mature pollen of Zea mays. Expression of partial cDNA of zm401 in maize and ectopic expression of zm401 in tobacco suggested it may play a role in anther development. Here we present the expression and functional characterization of this pollen specific gene in maize. Zm401 is expressed primarily in the anthers (tapetal cells as well as microspores) in a developmentally regulated manner. That is, it is expressed from floret forming stage, increasing in concentration up to mature pollen. Knockdown of zm401 significantly affected the expression of ZmMADS2, MZm3-3, and ZmC5, critical genes for pollen development; led to aberrant development of the microspore and tapetum, and finally male-sterility. Zm401 possesses highly conserved sequences and evolutionary conserved stable RNA secondary structure in monocotyledon. These data show that zm401 could be one of the key growth regulators in anther development, and functions as a short-open reading-frame mRNA (sORF mRNA) and/or noncoding RNA (ncRNA).

Abstract: Sphingolipids are important signaling molecules involved in various cellular activities. De novo sphingolipid synthesis is initiated by a rate-limiting enzyme, serine palmitoyltransferase (SPT), a heterodimer consisting of LONG-CHAIN BASE1 (LCB1) and LCB2 subunits. A mutation in the Arabidopsis thaliana LCB1 gene, lcb1-1, was found to cause embryo lethality. However, the underpinning molecular and cellular mechanisms remain largely unclear. Here, we report the identification of the fumonisin B1 resistant11-2 (fbr11-2) mutant, an allele of lcb1-1. The fbr11-2 mutation, most likely an allele stronger than lcb1-1, was transmitted only through female gametophytes and caused the formation of abortive microspores. During the second pollen mitosis, fbr11-2 initiated apoptotic cell death in binucleated microspores characteristic of nuclear DNA fragmentation, followed by cytoplasm shrinkage and organelle degeneration at the trinucleated stage. In addition, a double mutant with T-DNA insertions in two homologous LCB2 genes showed a phenotype similar to fbr11-2. Consistent with these observations, the FBR11/LCB1 expression was confined in microspores during microgametogenesis. These results suggest that SPT-modulated programmed cell death plays an important role in the regulation of male gametophyte development.

Abstract: Lectin receptor-like kinases (Lectin RLKs) are a large family of receptor-like kinases with an extracellular legume lectin-like domain. There are approximately 45 such receptor kinases in Arabidopsis thaliana. Surprisingly, although receptor-like kinases in general are well investigated in Arabidopsis, relatively little is known about the functions of members of the Lectin RLK family. A number of studies implicated members of this family in various functions, such as disease resistance, stress responses, hormone signaling, and legume–rhizobium symbiosis. Our current work demonstrated that mutation in one Lectin RLK gene led to male sterility in Arabidopsis. The sterility was due to defects in pollen development. Pollen development proceeded normally in the mutant until anther stage 8. After that, all pollen grains deformed and collapsed. Mature pollen grains were much smaller than wild-type pollen grains, glued together, and totally collapsed. Therefore, the mutant was named sgc, standing for small, glued-together, and collapsed pollen mutant. The mutant phenotype appeared to be caused by an unidentified sporophytic defect due to the mutation. As revealed by analysis of the promoter-GUS transgenic plants and the gene expression analysis using RT-PCR, the gene showed an interesting temporal and spatial expression pattern: it had no or a low expression in young flowers (roughly before anther stage 6), reached a maximum level around stages 6–7, and then declined gradually to a very low level in young siliques. No expression was detected in microspores or pollen. Together, our data demonstrated that SGC Lectin RLK plays a critical role in pollen development.

Abstract: The inaccessibility of the zygote and proembryos of angiosperms within the surrounding maternal and filial tissues has hampered studies on early plant embryogenesis. Somatic and gametophytic embryo cultures are often used as alternative systems for molecular and biochemical studies on early embryogenesis, but are not widely used in developmental studies due to differences in the early cell division patterns with seed embryos. A new Brassica napus microspore embryo culture system, wherein embryogenesis highly mimics zygotic embryo development, is reported here. In this new system, the donor microspore first divides transversely to form a filamentous structure, from which the distal cell forms the embryo proper, while the lower part resembles the suspensor. In conventional microspore embryogenesis, the microspore divides randomly to form an embryonic mass that after a while establishes a protoderm and subsequently shows delayed histodifferentiation. In contrast, the embryo proper of filament-bearing microspore-derived embryos undergoes the same ordered pattern of cell division and early histodifferentiation as in the zygotic embryo. This observation suggests an important role for the suspensor in early zygotic embryo patterning and histodifferentiation. This is the first in vitro system wherein single differentiated cells in culture can efficiently regenerate embryos that are morphologically comparable to zygotic embryos. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during the early stages of plant embryogenesis.

Abstract: Ogura cytoplasmic male sterility (CMS) occurs naturally in radish and has been introduced into rapeseed (Brassica napus) by protoplast fusion. As with all CMS systems, it involves a constitutively expressed mitochondrial gene which induces male sterility to otherwise hermaphroditic plants (so they become females) and a nuclear gene named restorer of fertility that restores pollen production in plants carrying a sterility-inducing cytoplasm. A correlative approach using light and electron microscopy was applied to define what stages throughout development were affected and the subcellular events leading to the abortion of the developing pollen grains upon the expression of the mitochondrial protein. Three central stages of development (tetrad, mid-microspore and vacuolate microspore) were compared between fertile, restored, and sterile plants. At each stage observed, the pollen in fertile and restored plants had similar cellular structures and organization. The deleterious effect of the sterility protein expression started as early as the tetrad stage. No typical mitochondria were identified in the tapetum at any developmental stage and in the vacuolate microspores of the sterile plants. In addition, some striking ultrastructural alterations of the cell's organization were also observed compared with the normal pattern of development. The results showed that Ogu-INRA CMS was due to premature cell death events of the tapetal cells, presumably by an autolysis process rather than a normal PCD, which impairs pollen development at the vacuolate microspore stage, in the absence of functional mitochondria.

Abstract: A major factor affecting spring canola (Brassica napus) production in Canada is killing frosts during seedling development in the spring and seed maturation in the fall. The objective of this study was to explore the possibility of producing spring canola lines with mutations that have altered biochemical pathways that increase cold tolerance. The approach was to generate UV point mutations in cultured microspores followed by chemical in vitro selection of individual mutant microspores or embryos resulting in measurable alterations to various biochemical pathways with ele- vated levels of key defense signaling molecules such as, salicylic acid (SA), p-Fluoro-D,L-Phenyl Alanine (FPA), and jasmonic acid (JA). In addition, since proline (Pro) is known to protect plant tissues in the cold-induced osmotic stress pathway, mutants that overproduce Pro were selected in vitro by using three Pro analogues: hydroxyproline (HP), azetidine-2-carboxylate (A2C); and, 3,4-dehydro-D,L-proline (DP). Of the 329 in vitro selected mutant embryos produced, 74 were identified with significant cold tolerance compared to their donor parents through indoor freezer tests at -6� C, and 19 had better winter field survival than winter canola checks. All chemically selected mutant doubled haploids with increased cold tolerance compared well with parent lines for all seed quality and agronomic parameters.

Abstract: The aim of this study was the improvement of embryo production in wheat anther culture. Three butanol alcohols, n-butanol, sec-butanol and tert-butanol, were evaluated for their effect on microspore embryogenesis in two spring cultivars of wheat, Pavon and Caramba. Application of n-butanol, at 0.1 and 0.2% (v/v) in the induction media for 5 h, highly improved embryo production in both cultivars. Sec- and tert-butanol performed similarly to control plates. Regeneration ability was unaffected by any butyl-alcohol treatment. As a consequence of the higher embryo production after n-butanol treatment, the number of green regenerated plants increased up to five times in cultivar Pavon and up to three times in cultivar Caramba. The percentage of green plants was improved or unaffected by the treatment. Doubled haploid plant production was between 2 and 4 times higher after n-butanol treatment than in control plates. Therefore, n-butanol was successfully applied in the production of wheat doubled haploids. This primary alcohol is known as an activator of phospholipase D and has been previously reported to disrupt cortical microtubules and detach them from the plasma membrane in plants. Its effects on androgenetic induction could confirm the importance of microtubule regulation in plant cell fate, specifically in microspore development. A possible implication of phospholipase D is discussed.

Abstract: A microspore mutagenesis protocol was developed for Brassica rapa, Brassica napus and Brassica juncea for the production of double haploid lines with novel fatty acid profiles in the seed oil Freshly isolated Brassica microspores were first cultured with ethyl methane sulphonate (EMS) for 15 h The EMS was removed and the microspores were then cultured according to the standard Brassica microspore culture protocol This protocol was used to generate over 80 000 Brassica haploid/double haploid plants Field evaluation of B napus and B juncea double haploids was conducted between 2000 and 2003 Fatty acid analysis of the B napus double haploid lines showed that saturated fatty acid proportions ranged from 50% to 77% For B juncea, saturate proportions ranged from 54% to 95% Of the 7000 B rapa lines that were analysed, 197 lines had elevated oleic acid (> 55%), 69 lines had reduced α-linolenic acid (<8%) and 157 lines had low saturated fatty acid proportions (<5%), when compared with the parental lines

Abstract: Unlike animals, whose gametes are direct products of meiosis, plant meiotic products undergo additional rounds of mitosis, developing into multicellular haploid gametophytes that produce egg or sperm cells. The complex development of gametophytes requires extensive expression of the genome, with DNA-dependent RNA polymerases I, II, and III being the key enzymes for nuclear gene expression. We show that loss-of-function mutations in genes encoding key subunits of RNA polymerases I, II, or III are not transmitted maternally due to the failure of female megaspores to complete the three rounds of mitosis required for the development of mature gametophytes. However, male microspores bearing defective polymerase alleles develop into mature gametophytes (pollen) that germinate, grow pollen tubes, fertilize wild-type female gametophytes, and transmit the mutant genes to the next generation at moderate frequency. These results indicate that female gametophytes are autonomous with regard to gene expression, relying on transcription machinery encoded by their haploid nuclei. By contrast, male gametophytes make extensive use of transcription machinery that is synthesized by the diploid parent plant (sporophyte) and persists in mature pollen. As a result, the expected stringent selection against nonfunctional essential genes in the haploid state occurs in the female lineage but is relaxed in the male lineage.

Abstract: In our earlier work, a pollen-expressed polygalacturonase gene BcMF6 was isolated from floral bud of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) by cDNA-amplified fragment length polymorphism (cDNA-AFLP) transcript profiling and rapid amplification of cDNA ends (RACE). To unravel the biological function of BcMF6 gene, the antisense fragment from the BcMF6 gene with A9 promoter and CaMV35S promoter was transferred into flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis). Out of transgenic plants transformed with the antisense gene constructed from the BcMF6, transgenic line with A9 promoter have a similar appearance to that with CaMV35S promoter. Morphological investigations showed that the transgenic plants developed the smaller floral organ with thin anther and less pollen. Pollen germination test indicated that only near 50% the pollen from the transgenic line could normally germinate. Further scanning electron microspore analysis of transgenic plants confirmed that half of pollen was abnormal. Cytological comparisons of microspore development also demonstrated that process of microsporogenesis was held up, microspores maturation was disrupted and pollen grain fail to separate, finally. In a word, the present study revealed that BcMF6, as a polygalacturonase gene, has a role in pollen maturation and pollen tube growth.

Abstract: Anther culture was used to obtain dihaploid (DH) coconut plants and their ploidy level was determined by flow cytometric analysis. Simple sequence repeat (SSR) marker analysis was conducted to identify the homozygous diploid individuals. Ploidy analysis showed that 50% of the tested plantlets were haploid and 50% were diploid. Polymorphic fragments of the mother palm and their segregation patterns in anther-derived plantlets were used to determine the origin of the diploid plantlets. Using a diagnostic SSR marker (CNZ43), all the diploid plantlets tested were identified as being derived from microspores (i.e. were homozygous) and were thus candidates for use in coconut breeding programs.

Abstract: Three Indian Brassica juncea cultivars were studied for embryogenic response of microspores, microspore embryo regeneration, ploidy assessment of microspore-derived plants and their diploidization. Genotype dependence for microspore totipotency was observed and a significant effect of genotype by bud size selection was established. The addition of activated charcoal in NLN medium containing 13% (w/v) sucrose and 10 μM silver nitrate resulted in a fourfold increase in microspore embryogenesis, ranging from 100 to 405 embryos per Petri dish corresponding to 2,700–10,935 embryos per 100 buds. Conversion/germination of embryos produced in presence or absence of activated charcoal was similar but air-drying of microspore embryos was essential. Incubation of microspore embryos at 4 ± 1°C for 10 days in dark resulted in 82.3% conversion. The majority of plants produced from these embryos was haploid. Treating microspore-derived plants at the 3–4 leaf growth stage with 0.34% colchicine for 2–3 h resulted in greatest survival (70%) and chromosome doubling (75%) frequencies. Doubled haploid plants were self-pollinated and grown to maturity under field conditions.

Abstract: The switch of programme in barley anther culture is discussed with reference to the formation of green and albino plants. A theory of albinism is proposed based on the premise that at the critical microspore stage, plastids are in a state of metamorphosis preparatory to their role in the gametophyte generation. Pollen cells formed during the cold treatment which precedes culture of the anthers lack the usual gametophytic traits and have no divisional constraints, but they cannot develop green plastids because of deficiencies at the plastid level. Calluses derived from such cells give rise to albinos. Formation of green plants is equated with younger spores in which the two pollen cells are formed in culture rather than the pretreatment. Culture appears to have an important role in sporophytic expression apart from that in sustaining growth. Plastids in these younger spores are considered to be appropriate to greening, but may not be able to develop a photosynthetic apparatus because of deficiencies at the nuclear level. Formation of the pollen cells in culture, following the pretreatment, may be necessary for unmasking genes essential to greening. Ways of controlling the production of green plants at a level acceptable to breeders are suggested.

Abstract: Cabomba is a small water lily genus that is native to the New World. Studies of pollen development and associated changes in the anther yield valuable characters for considering the evolution of reproductive biology in seed plants. Here we characterized the complete ontogenetic sequence for pollen in Cabomba caroliniana. Anthers at the microspore mother cell, tetrad, free microspore, and mature pollen grain stages were studied using scanning electron, transmission electron, and light microscopy. Tetragonal and decussate tetrads both occur in C. caroliniana, indicating successive microsporogenesis. The exine is tectate-columellate, and the infratectal columellae are the first exine elements to form, followed by a continuous tectum and a thin foot layer. A lamellate endexine initiates in the early free microspore stage, but becomes compressed in mature grains. Tectal microchannels and sculptural rods also initiate during the early free microspore stage, and significant pollenkitt deposition follows, supporting the hypothesis that these elements function in entomophily. The tapetum is morphologically amoeboid, with migratory tapetal cells directly contacting developing free microspores within the anther locule. Results from this study illustrate the importance of including ontogenetic data in analyzing pollen characters and in developing evolutionary and ecological hypotheses. The new palynological data also emphasize the character plasticity that occurs in basal angiosperms.

Abstract: The effect of ZnSO4 concentration on barley (Hordeum vulgare L.) microspore embryogenesis was investigated using cultivars of different androgenetic response. Concentrations from 0 (control) to 600 μM in the stress pre-treatment medium alone or in combination with 30 (control) to 600 μM in the embryo induction medium were assayed in anther culture. Incorporation of Zn2+ in the pre-treatment medium itself did not affect microspore embryogenesis. The optimum concentration in the stress pre-treatment and induction media was 180 μM for cultivars (cvs.) Igri and Reinette, and 90 μM for cv. Hop. A significant increase of 30 and 300% in cv. Igri and Reinette, respectively, were produced with 180 μM ZnSO4 in both the number of embryos and green plants. In order to confirm the effect of Zn2+ on microspore embryogenesis this micronutrient was incorporated in the induction medium of isolated microspore cultures of cv. Igri. Concentrations of 90–300 μM ZnSO4 resulted in an increase of 40–53% in the number of embryos and green plants. All these results indicate that the beneficial effect of Zn2+ is exerted mainly during the culture phase, increasing the number of embryos, leading to an increased number of green plants, but it had no effect on percentage of regeneration or green plants.

Abstract: Haploid microspore-derived embryos (MDEs) constitute a unique material for the introduction of new traits into winter oilseed rape (Brassica napus). MDEs have been transformed by usingAgrobacterium tumefaciens strains EHA105 and LBA4404, both carrying the binary vector pKGIB containing theuidA gene encoding β-glucuronidase (GUS) and thebar gene as a marker of resistance to phosphinotricin. Transformed embryos expressed GUS and regenerated plants that were resistant to herbicide Basta, as confirmed by a leaf-painting test. Progeny plants of the transformant T-39 were all transgenic, as they inherited T-DNA from their doubled haploid parental plant. Southern-blot analysis confirmed the integration and transmission of T-DNA into T1 plants. Transformation of MDEs facilitates the obtaining of winter oilseed rape homozygous for the introduced genes.

Abstract: Haploid induction in rocket (Eruca sativa Mill.), a novel and increasingly important vegetable, was studied in microspore culture. A procedure based on a high sucrose NLN medium and heat shock treatment resulted in nuclear divisions and embryo induction. The effect of genotype both among seed lots and among single plants was a major factor influencing embryo formation. The addition of activated charcoal was essential for obtaining reproducible results, 0.2 mg l−1 being superior to 1.0 mg l−1. A 24 h heat shock treatment at 32°C doubled the embryogenic response compared to a 48 h treatment. Embryo conversion was only efficient (23%) for embryos that had been cultured on medium with activated charcoal and subcultured on solid B5 medium; pretreatment of embryos with ABA or desiccation for 1–3 weeks inhibited embryo conversion. Analysis of ploidy level revealed that the majority (65.6%) of 489 regenerated plantlets tested were diploid. Breeding programs and genetic studies of rocket are likely to benefit substantially from the established method.

Abstract: The nuclear localization of blue-staining flavanols was investigated histochemically throughout microsporogenesis in yellow cypress (Callitropsis nootkatensis (D. Don) Oerst., formerly Cupressus nootkatensis), juniper (Juniperus communis L.) and yew (Taxus baccata L.). During meiotic development, both the cytoplasm and nuclei of microspores of all species contained varying amounts of flavanols; however, the flavanols were largely confined to the nuclei in microspores just released from tetrads. Quantification by HPLC analysis indicated that, in all species, catechin and epicatechin were the dominant nuclear flavanols. At the early free microspore stage, the nuclear flavanols were barely detectable in all species, but they increased fivefold on incubation in the presence of 0.1 mM benzylaminopurine (BA) or zeatin. Histochemical studies revealed that, in addition to non-fluorescing flavanols, microspores contained yellow-fluorescing flavonoids, which yielded a distinct HPLC flavonoid profile for each species. In yellow cypress, the hydrolyzed flavonoids were identified as quercetin, apigenin, kaempferol and luteolin, whereas only quercetin and myricetin were found in microspores of juniper and in anthers of yew. Application of a UV-VIS titration technique revealed that the aglycone quercetin seems to interact more strongly with histone H3 than either glycoside rutin or kaempferol.

Abstract: Efforts were made to study microsporogenesis and genetics of fertility restoration of A(4) cytoplasmic-nuclear male-sterility (CMS) system in pigeonpea. The process of microsporogenesis in the male-sterile (ICPA 2039) and its maintainer (ICPB 2039) plants was normal up to the tetrad formation stage. The tapetal cells in the male-sterile anthers degenerated soon after tetrad formation, resulting in shriveled and degenerated microspores. In the maintainer plants, the tapetal cells were normal and microspores were functional. The breakdown of the tapetum before the completion of microsporogenesis was the major cause for the expression of male sterility in A(4) CMS system. The studies on the inheritance of fertility restoration showed that in 3 crosses, a single dominant gene; in 1 cross, 2 duplicate genes; and in another cross, 2 complimentary genes governed the fertility restoration.

Abstract: Comparative expression profiling of flower buds in two male sterile lines [genic male sterile (GMS) and cytoplasmic male sterile (CMS)] with its male fertile maintainer line in Chinese cabbage pak-choi was performed using cDNA-AFLP technology to identify the genes implicated in male sterility. A novel gene BcMF10, sharing high sequence similarity to the function-unknown DUF1216 family in Arabidopsis was isolated, whose expression was absent in the flower buds of the GMS and CMS lines but present in the male fertile maintainer line. Temporal and spatial expression pattern analysis revealed that BcMF10 began to be expressed in tapetal cells and microspores during meiosis. Expression in tapetal cells was persistent until the degeneration of tapetum, and expression in microspores reached a peak during the tetrad stage but gradually declined as development proceeded. RNA interference technology was used to address the biological function of BcMF10. The RNAi transgenic Chinese cabbage pak-choi lines showed normal vegetative growth and reproductive development, but poor pollen germination. Scanning electron microscopy (SEM) showed that most of the transgenic pollen was deformed and exhibited an irregular shape with an abnormal number and distribution of germinal furrows. It is speculated that BcMF10 may encode a protein that plays a role in the formation of intine wall.

Abstract: Anatomical changes occurring during the microsporogenic development of P. salicina Lindl. were studied in male fertile and male sterile genotypes. Male fertile pollen grains showed three well determined pore regions, without ektexine. Intine was thick and surrounded the vegetative cell. Vegetative cells enclosed the generative cells; their cytoplasm was rich in plastids, abundant RER and active mitochondria. Development of sterile pollen was different from the meiosis step. Microspores did not show germination pores and ektexine was continuous around the whole grain. Pollen grains showed an atypical shape. The tapetum persisted after the tetrad stage and showed hypertrophy and vacuole development, resulting in abnormal microspore development. Only a few pollen grains and rudiments of collapsed microspores close to the anther wall were formed at anthesis.

Abstract: Populus bolleana is a variety of P. alba, commonly used in poplar breeding programs in China. Developmental biology that involves staminate flowers, microsporogenesis and microgametogenesis of P. bolleana is essential for Populus improvement in cross breeding for better characteristics in sexual reproduction. Flower morphology and pollen development were described and illustrated using anatomical, sectioning and stain-clearing techniques. The results show that microsporocytes undergo a regular meiotic process, but some multi-nucleate microspores occur at the microspore stage. It takes five days for microsporocytes to develop to mature pollen by forcing flower branches under greenhouse conditions. Additionally, an important relationship was found between stages of meiosis and anther colors. Microspore tetrads formed when the anther color turned yellow, whereas, when the pollen matured, the anther was red and the tapetum degenerated completely. When mature pollen grains are formed, flower buds develop into male catkins. In the end, filament elongated and pollen grains were released from dehisced anthers.