Microtubule organization of in situ and isolated generative cells in Zephyranthes grandiflora Lindl

Summary Microtubule organization in the generative cells of Zephyranthes grandiflora was investigated by immunofluorescence microscopy with a monoclonal anti--tubulin. The experimental materials used were generative cells located within pollen grains and tubes (i.e., in situ) as well as those artificially isolated after osmotic shock or grinding treatments of the pollen grains. Diverse microtubule organization patterns were revealed. In situ, the generative cells appeared spindle-shaped and contained mainly longitudinally oriented microtubule bundles, although other types were found as well. After isolation, as the alteration in microtubule patterns took place, the spindle-shaped generative cells became ellipsoidal and then spherical. In the ellipsoidal cells a transitional form consisting of a mixture of microtubule bundles and meshes could be found. In spherical cells the mesh structure appeared to be the predominant pattern. These results indicate that the microtubule cytoskeleton of the generative cells can change easily from one structural form to another in accordance with environmental conditions and may play an important role in determining the cell shape.     

Microtubule changes during the development of generative cells in Hippeastrum vittatum pollen

Summary The three-dimensional organization of microtubules in generative cells during their development in pollen grains of Hippeastrum vittatum and the dynamic changes that occur were studied by collecting large quantities of fixed and isolated generative cells for immunofluorescence microscopy. The framework configuration and the arrangement pattern of the microtubule organization was investigated. The microtubule framework changed in shape from being spherical at an early stage to being long spindle-shaped at maturity: various transitional forms were observed: ellipsoidal, pear-shaped and short spindle-shaped. The microtubule arrangement making up this framework changed correspondingly from the original network, which was random in distribution, to axially oriented long bundles via an intermediate pattern composed of a mixture of networks with long bundles. However, cells with the same framework configuration might be heterogeneous in microtubule arrangements.     

Specific phosphoproteins in the initial period of tobacco pollen embryogenesis

Several phosphoproteins specifically correlated with the induction of embryogenic cells were detected in immature pollen grains of Nicotiana tabacum L. By regulating the concentration of glutamine in the medium the developmental pathways of immature pollen grains isolated at the mid-bicellular stage could be controlled, resulting in the formation of either mature pollen grains or embryogenic cells. Different phosphoproteins, designated as a-d and as e-i, respectively, were detected when the pollen grains either became embryogenic cells in glutamine-free medium, or when they were allowed to mature in glutamine-containing medium. The formation of embryogenic cells was suppressed by adding glutamine or cytokinin to the glutamine-free medium, nor did it occur with pollen grains at younger or older stages, and in these cases the phosphoproteins a-d were detectable only partially or faintly. The phosphoproteins a-d and e-i thus may be one of the factors necessary to direct the developmental pathway of immature tobacco pollen grains to embryogenic cells and to mature pollen grains, respectively.The authors thank Dr. V.S. Jaiswal (Botany Department, Banaras Hindu University, Varanasi, India) for his valuable suggestion in the preparation of the paper. This work was supported by a Grantin-Aid for special project research from the Ministry of Education, Science and Culture of Japan.     

Localization and release of allergens from tapetum and pollen grains ofBetula pendula

Summary Although intact pollen grains are assumed to be the primary carrier of pollen allergens, specific immunoreactive components have been found in other aerosol fractions, e.g., starch grains and remains of tapetal cells Cryo-scanning-electron-microscopy results demonstrate the presence of a clear network of strands connecting the tapetum with the microspores. The distribution of protein in tapetal orbicules, pollen wall, and pollen cytoplasm was tested by histochemical stains for light microscopy and transmission electron microscopy. The protein is mainly localized at the apertures and starch grains in the cytoplasm of pollen and in the core and on the surface of tapetal orbicules. Monoclonal antibodies Bv-10, BIP3, and BIP4 have been used to locate the cellular sites of pollen and tapetal allergens inBetula pendula (syn.B. verrucosa). The application of rapid-freeze fixation prevented relocation of allergens from their native sites. The allergens are predominantly found in the starch grains and to lesser extent in the exine. We also tested interactions between mature birch pollen and human fluids: saliva, nostrils fluid, and eyes solution. The aim was to mimic more closely the in vivo situation during allergenic response. In all cases we observed several pollen grains that were burst and had released their cytoplasmic contents. In the nose the allergens are released from the pollen within minutes. In rhinitis, nasal pH is increased from the normal pH 6.0 to 8.0. When we used nasal fluid at pH 8.0, the number of ruptured pollen grains increased. The mechanism that might induce formation of small allergen-bearing particles from living plant cells is discussed.     

The Effects of Air Pollution on Structures, Proteins and Allergenicity of Pollen Grains

The prevalence of allergic disease has increased world wide during the last decades. Pollen allergy is the most typical form of allergic disease. The increase in its frequency during recent years is the most evident. Environmental factors play an important role in the problem of pollen allergy in large cities. The aim of this research is determination of allergenicity of Canna pollen in polluted and non-polluted conditions, detection of their allergenic proteins and also elucidation of some microscopic effects of air pollutants on pollen structure and proteins. Mature and immature pollen grains of Canna indica were collected from polluted and non-polluted areas. Pollen grains were studied by scanning electron microscopy. Mice were sensitized by injection of pollen extract and an adjuvant for five times. Allergy potency of different pollen extracts were compared by means of skin test, blood eosinophills number and IgE levels in sensitized and treated animals. Pollen proteins were studied by SDS-PGE and allergenic proteins were detected by immunoblotting techniques. Scanning electron microscope study of the pollen grains showed that in polluted areas, air born particles accumulated on the surface of pollen and changed both pollen's shape and pollen's tectum. Also many vesicles were released out of polluted pollen and the pollen material agglomerated on the surface of pollen. SDS-PAGE showed that different proteins exist in mature and immature pollen. In pollen collected from polluted area, some of protein bands between 22 and 45 kDa were disappeared . Also in all polluted pollen grains, protein content of pollen decreased in response to air pollution causing the release of pollen proteins. According to our experiments and regarding induction of allergic symptoms, the polluted pollen is more effective than non-polluted one, and mature pollen has more allergy potency than immature one.     

Microsporogenesis of the feathers (fertile branches derived from annual buds) in Vitis vinifera,cv Picolit giallo

Abstract

Using light and electron microscopy, we have studied the microsporogenesis and tapetal development of the feathers in two different low producing clones of Picolit giallo (sp. Vitis vinifera). In these clones while the productivity of the main branches (fertile branches originated from buds, formed in the previous year, that remained silent during the winter) is very low, that of the feathers (fertile branches derived from annual buds) is always normal.

The microsporogenesis and tapetal development proceed normally in almost all the examined anthers; it is remarkable that at the tetrad stage the tapetal cells appear well structured without any degeneration symptom, unlike what observed for the main branches. Moreover in most of the mature anthers the pollen grains are numerous, pleinty of organelles and show sometimes thickenings in the callose layer under their wall. The tapetal cells of these anthers have disappeared. Only in few anthers we observed the presence of collapsed pollen grains and tapetal cells with anomalous development, that are still present when the pollen grains are mature. This rare situation for the feathers is on the contrary frequent for the main branches.     

Scanning and transmission electron microscopy related to immunochemical analysis of grass pollen

The exine stereostructure (scanning electron microscopy) and ultrastructure (transmission electron microscopy) of pollen of seven grass species, is related to the allergens extracted from these pollen grains. The heterogeneity of the allergens was studied by the immunoprint technique and revealed by labelling the binding of grass pollen sensitive patients IgE antibodies. Using patient sera recognizing a very restricted number of allergens, we showed that a group of pollen had a great number of allergens in common (Dactylis, Agrostis, Festuca, Lolium, Holcus) and, in decreasing cross reactivities order, we found Avena and, finally, Zea mays. The tectum stereostructure shows presence of insulae in all pollen grains except in Zea mays which has small isolated spinules. These insulae are separated by very wide and deep interinsular spaces in Avena sativa with connections between insulae. In the remaining species, no connections were seen between the insulae. These observations were in good correlation with the immunological cross-reactivity of the allergens present in the pollen. In all species, there are microperforations in the bottom of the interinsular spaces, which are the opening of the tectal microchannels.     

Ultrastructure of the sperms ofPlumbago zeylanica

Summary Male gametes ofPlumbago zeylanica were examined in pollen grains and tubes using light and electron microscopy of chemically and physically fixed tissues, and Nomarski interference microscopy of isolated, living sperm cells. Male gametes are elongate, spindleshaped cells containing a nucleus, mitochondria, ER, ribosomes, vesicles, dictyosomes, probable microfilaments, and a variable number of plastids. In mature pollen grains ofP. zeylanica, the two sperm cells are directly linked; they share a transverse cell wall with plasmodesmata and are enclosed together by the inner vegetative cell plasma membrane. One of these two sperms is also associated with the vegetative nucleus as a consistent feature of pollen grain organization. The basis of this association appears to be a long, narrow projection of the sperm cell (averaging < 1 m wide and about 30 m long) which wraps around the periphery of the vegetative nucleus and occupies embayments of that nucleus. This association is maintained throughout pollen tube growth but becomes less extensive near the completion of tube growth and is severed following tube discharge. The consistent occurrence of the sperm-vegetative nucleus association in pollen grains, tubes and isolated pollen cytoplasm suggests that the two structures may be directly connected, but attempts to visualize this type of connection were unsuccessful. Possibly, the entwining nature and extent of complementary interfaces between vegetative nucleus and sperm may have a role in stabilizing their association. Functionally, the two sperms and vegetative nucleus appear to travel as a linked unit within the pollen tube, possibly increasing the effectiveness of gamete delivery and helping to ensure nearly simultaneous transmission of sperms into the receptive megagametophyte.     

Pollen morphology of Quercus sect. Ilex and its relevance for fossil pollen identification in southwest China

Pollen grains of 16 modern species of Quercus were examined by scanning electron microscopy (SEM) and light microscopy (LM). The focus of this investigation was on a small group (c. eight species) of alpine, sclerophyllous oaks that may form a monophyletic group within sect. Ilex. The data were combined with published data for Polar axis (P) and Equatorial diameter (E) for more than 80 species. Based on pollen morphology Quercus pollen grains were divided into three types: (1) The Cyclobalanopsis type is smallest (generally < 23 μm) in size, with P/E ratios of approximately 1.08 and a psilate or fine-grained surface; (2) Quercus sect. Ilex type is 23–29 μm, the P/E ratio is approximately 1.24 (prolate) and it has a rugulate pollen surface; (3) the Quercus sect. Quercus type is generally > 29 μm in size, has a P/E ratio of approximately 1.04, and a clearly verrucate pollen surface. Based on a comparison with modern pollen size and ornamentation, fossil pollen grains of the Quaternary Qinghai Lake core can be assigned to the Chinese southwest mountain oak group of Quercus sect. Ilex (in particular Quercus semecarpifolia). In addition, we show that the pollen percentages of Quercus rapidly increased to the highest level during the glacial period. Using SEM for the identification of fossil Quercus pollen grains is essential to ensure the accuracy of differentiating evergreen Quercus types (sect. Ilex versus sect. Cyclobalanopsis) during the last glacial period in southwest China.     

Electrorotation of Isolated Generative and Vegetative Cells, and of Intact Pollen Grains of Lilium longiflorum

The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m⁻¹. The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques. Received: 9 June 1997/Revised: 4 August 1997     

玉米生活精子的分离及扫描电镜观察(简报)

生殖细胞的分离和纯化是植物生殖生物学及生殖细胞工程的发展基础之一。70年代,Cass(1973)首次从大麦花粉管及花粉粒中分离精子;到80年代后期,分离精子已成为国际上实验生殖生物学中的热点。由于大量分离和纯化的技术进一步完善,已从一些植物的     

The unique pollen morphology of Duparquetia (Leguminosae: Caesalpinioideae): developmental evidence of aperture orientation using confocal microscopy

BACKGROUND AND AIMS: The phylogenetic affinities of the aberrant monotypic genus Duparquetia (subfamily Caesalpinioideae) are at present unresolved. Preliminary results from molecular analyses suggest a basal, isolated position among legumes. A study of Duparquetia pollen was carried out to provide further morphological characters to contribute to multi-data set analyses. Understanding the development of Duparquetia pollen was necessary to clarify the orientation of the apertures. METHODS: Pollen grains and developing microspores were examined using light microscopy, confocal microscopy and scanning electron microscopy. Evidence for the orientation of the apertures was provided by the examination of microspores within developing tetrads, using (a) confocal microscopy to locate the position of the ectoapertures, and (b) light microscopy and Alcian blue stain to locate the position of the endoapertures. KEY RESULTS: Confocal microscopy has been used for the first time to examine developing microspores in order to obtain information on ectoapertures that was unavailable using other techniques. Pollen in Duparquetia develops in tetrahedral tetrads as in other eudicots, with the apertures arranged in a modified pattern following Fischer's rule. Pollen grains are asymmetrical and have one equatorial-encircling ectoaperture with two equatorial endoapertures, a unique feature in Leguminosae, and in eudicots. CONCLUSIONS: The pollen morphology of Duparquetia is so unusual that it provides little information to help determine its closest relatives. However, it does fit with a pattern of greater pollen morphological diversity in the first-branching caesalpinioid legume groups than in the more derived clades. The latitudinal ectoaperture of Duparquetia is unique within the Fabales and eudicot clades, resembling more closely the monosulcate pollen found in monocots and basal angiosperms; however, developmental patterns are recognizably similar to those of all other legume pollen types.     

Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells ofPelargonium zonale during pollen development

Summary In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system     

Some permeability properties of angiosperm pollen grains,pollen tubes and generative cells

Summary The permeability of pollen grains, pollen tubes and generative cells of Helleborus foetidus and Galanthus nivalis has been investigated using four probes spanning a wide range of molecular weights: 4,6-diamidino-2-phenyl indole (DAPI; mol.wt. 350). Evans blue (mol.wt. 960), FITC-dextran (average mol.wt. 19400) and FITC-albumin (average mol.wt. 67000). DAPI penetrated into the vegetative cells of desiccated and hydrated pollen, and also entered growing pollen tubes. In contrast, the generative cells of hydrated pollen and of pollen tubes were highly resistant to penetration, as they were when isolated in osmotically balancing medium. Evans blue failed to enter intact generative cells under any of the conditions tested. The dye ultimately entered the vegetative cells of some pollen grains, but these were non-germinable. Growing pollen tubes invariably resisted penetration. Neither of the high molecular weight conjugates entered germinable pollen grains or intact pollen tubes. The results suggest that it is highly unlikely that DNA fragments of high molecular weight can enter viable pollen, pollen tubes or generative cells under any normal conditions.     

A palynological study of the genus Mentha L. (Lamiaceae)

The pollen morphology and exine structure of 10 Mentha L . species were investigated using light microscopy and scanning electron microscopy. The pollen grains of all 11 species were hexazonocolpate with granular membranes and a circular amb, varying in shape from prolate-spheroidal to suboblate. Different colpus shapes were recognized in M . ×  dumetorum . The exine was bireticulate in section Pulegium , and reticulate in section Menthae . A correlation was found between pollen size and chromosome number. The results indicate that the pollen characters of the genus Mentha are valuable for taxonomic applications and may be useful for classification.  © 2008 Uludag University. Journal compilation © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 141–154.     

Development of a semi-automatic system for pollen recognition

A semi-automatic system for pollen recognitionis studied for the european project ASTHMA. The goal of such a system is to provideaccurate pollen concentration measurements. This information can be used as well by thepalynologists, the clinicians or a forecastsystem to predict pollen dispersion. At first,our emphasis has been put on Cupressaceae, Olea, Poaceae and Urticaceae pollen types. The system is composed of two modules: pollengrain extraction and pollen grain recognition. In the first module, the pollen grains areobserved in light microscopy and are extractedautomatically from a pollen slide coloured withfuchsin and digitized in 3D. In the secondmodule, the pollen grain is analyzed forrecognition. To accomplish the recognition, itis necessary to work on 3D images and to usedetailed palynological knowledge. Thisknowledge describes the pollen types accordingto their main visible characteristerics and tothose which are important for recognition. Somepollen structures are identified like the porewith annulus in Poaceae, the reticulum in Oleaand similar pollen types or the cytoplasm inCupressaceae. The preliminary results show therecognition of some pollen types, likeUrticaceae or Poaceae or some groups of pollentypes, like reticulate group.     

Floral micromorphology and microsporogenesis of the gynodioecious herb Glechoma longituba (Lamiaceae)

Gynodioecy, the phenomenon of having both hermaphrodite and female (i.e. male‐sterile) individuals within the same population, is an important intermediate step in the evolution of separate sexes in flowering plants. In this study, we investigated the floral micromorphology and microsporogenesis of the gynodioecious herb Glechoma longituba from four natural populations in Korea. The floral micromorphological characters of the different sex morphs were examined and compared using scanning electron microscopy (SEM), and the ultrastructure of microspores during microsporogenesis was studied. We also examined the development of anthers and pollen grains in the three sexual morphs (i.e. hermaphrodites, females, and gynomonoecious, i.e. individuals with a mixture of female and hermaphroditic flowers) by embryological investigation. The major difference in anther development between the three phenotypes was the early disintegration of the tapetal cells in the anthers of female flowers. While mature fertile pollen grains were found in both hermaphrodite and gynomonoecious phenotypes, females did not produce any pollen grains. In addition, both fertile and sterile pollen grains in gynomonoecious phenotypes were frequently observed. The results of the present study indicate that floral micromorphological characters were not distinct between sexual morphs of G. longituba, except for the structure of the inner cell surfaces of the anther. The observed tapetum abnormalities and degeneration of pollen grains in both gynomonoecious phenotypes and females may be the consequence of inbreeding depression in hermaphrodites.     

The timetable for development of maternal tissues sets the stage for male genomic selection in Betula pendula (Betilaceae)

The influence of the sequence of maternal tissue development in Betula pendula upon the potential for male gamete selection was investigated, and the timing of the fixed abortion of one of the two ovules was determined. We used scanning electronic microscopy, confocal laser scanning microscopy, and blue light microscopy. The stigmas remain fresh throughout male anthesis, and may also last after its end, depending on ambient temperatures. The presence of germinated pollen does not induce stigmatic necrotization, and grains may arrive at different times. The pollen tube tips remain within the stigma base until the end of female anthesis. The ovules will not develop until after necrotization of the stigmas. The pollen tubes thus have a fair start to the ovules, regardless of their different arrival times and of the original positions of the pollen grains at the stigma surface. Therefore, competition among different microgametophytes is possible, in spite of low pollination intensity. Our results indicate that when the first pollen tube penetrates an ovule, this ovule starts to outgrow the other one, and even if the other is also penetrated, its vascular support soon atrophies and the megagametophyte will shrivel. Fertilization of both ovules was never seen in this study.     

Partner limitation and restoration of sexual reproduction in the clonal dwarf shrubLinnaea borealis L. (Caprifoliaceae)

Summary Reports suggest that there is widespread reproductive failure inLinnaea borealis in Britain, Scandinavia, and North America. Our investigations of Scottish populations of this clonal dwarf shrub indicate that, although visited by a number of different insects, pollen transfer in this species is highly effective and principally occurs by small flies (Muscidae). However, natural levels of fruit set varied between populations (from 0% to 25.1%) and reproductive failure was most severe in populations which were composed of single clones. Microscopic examination of stigmas showed that there is no barrier to pollen flow since at least 85% of stigmas have sufficient germinating pollen to effect seed set. Fluorescence microscopy of germinating pollen grains indicates high levels of pollen rejection in the style and only a small proportion of the pollen tubes were able to reach the ovary. It was concluded that lack of xenogamous pollination limits fruit formation in populations ofL. borealis. Reproductive success in an isolated population with extreme reproductive failure was restored by experimental field pollination with viable pollen imported from plants from another population. In Scotland,L. borealis occurs in small, isolated populations and restoration of reproduction can be achieved by the reintroduction of compatible mating partners. This is an important consideration for the conservation management of isolated populations since their long-term recovery may only be possible by translocation of different genotypes from elsewhere into the population. Habitat fragmentation in any part of the species range may impose a potential reproductive bottleneck by causing loss of population diversity and this could explain the low levels of seed set recorded for this species in other parts of its range.     

Morphological and functional characterization of BcMF13 in the antisense-silenced plants of Brassica campestris ssp. chinensis var. parachinensis

The gene Brassica campestris male fertility 13 (BcMF13, GenBank accession number EF158459) was isolated as a reproductive organ-specific gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis). It is exclusively expressed in stage four and five flower buds of fertile lines and is most strongly expressed in stamens. Here, we report a functional characterization of this BcMF13 gene in the antisense-silenced plants. The inflorescence of the BcMF13 mutant was compacted with anthers curved outside. The fertility of this mutant was greatly reduced with less than 5 seeds per silique. Under scanning electron microscopy, the mutant demonstrated numerous shriveled pollen grains with deep invaginations. The frequency of normal pollen grains was just 45.34%. The pollen mother cell, the tetrad, and the mature pollen of the BcMF13 mutant were abnormal resulting in the poor pollen vitality. Germination test in vivo suggested BcMF13 delayed the pollen tubes’ extension in the style. All these indicated BcMF13 had a vital role in pollen development of Chinese cabbage.