Single Pollen Grains Research Articles

Pollen Grain Size, Density, and Settling Velocity for Palmer Amaranth (Amaranthus palmeri)

Palmer amaranth is resistant to several herbicides, including glyphosate, and there is concern that the resistance traits can be transferred between spatially segregated populations via pollen movement. The objective of this study was to describe the physical properties of Palmer amaranth pollen, specifically size, density, and settling velocity (Vs), that influence pollen flight. The mean diameter for Palmer amaranth pollen, as determined by light microscopy, was 31 µm (range of 21 to 38 µm); mean pollen diameter as measured with the use of an electronic particle sizer was 27 µm (range of 21 to 35 µm). The mean density of the solid portion of the pollen grain was 1,435 kg m−3. Accounting for the density of the aqueous fraction, the mean density of a fully hydrated pollen grain was 1,218 kg m−3. By Stokes's law, the estimated mean theoreticalVsfor individual Palmer amaranth pollen grains was 3.4 cm s−1for the range of pollen diameters with a mean of 31 µm and 2.6 cm s−1for the range of pollen diameters with a mean of 27 µm. Results from laboratory studies indicated the majority of single pollen grains settled at a rate of 5.0 cm s−1. The difference between the theoretical and empirical estimates ofVswas likely due to changes in pollen density and shape postanthesis, which are not accounted for using Stokes's law, as well as the presence pollen clusters.

Germination and growth of sponge gourd ( Luffa cylindrica) pollen tubes and FTIR analysis of the pollen tube wall

Emergence of multiple pollen tubes from single pollen grains occurred both in vitro and in vivo in sponge gourd ( Luffa cylindrica (L.) Roem). The frequency with which pollen grains produced multiple pollen tubes in vivo (7.2%) was lower than that under in vitro conditions (14.9%). In pollen grains germinated in vitro, the total length of the multiple pollen tubes was greater than that of single pollen tubes, but individual tubes among the multiple tubes did not reach the same length as single tubes. Moreover, the growth of the single pollen tubes continued for a longer period in vitro than that of the multiple tubes. Fluorescence microscopy showed that callose was present throughout the pollen tube wall except in the apical part of growing pollen tubes, and nuclei moved into the longest of the multiple tubes. Results of Fourier transform infrared microspectroscopy indicated that abnormal cell wall components (peaks at 800–1000 cm −1) were more frequent in multiple pollen tubes lacking nuclei, and the pectin content (1733 cm −1) in multiple pollen tubes was much lower than that in single pollen tubes. These findings suggested that there were significant differences in pollen tube growth and wall composition between single and multiple pollen tubes, and that multiple pollen tubes had much less opportunity than single pollen tubes to reach the embryo sac and achieve double fertilization.

Characterization of Ragweed Pollen Adhesion to Polyamides and Polystyrene Using Atomic Force Microscopy

Pollen is a leading contributor to asthma and allergies, yet pollen adhesion to common indoor surfaces is not well understood. We report the adhesive behavior of short ragweed (A. artemisiifolia) pollen grains with Nylon 6 (N6) and Nylon 6,6 (N66), chosen due to their use in synthetic carpet, and three control surfaces: polyamide 12 (PA12), polystyrene (PS), and silicon. The forces were measured by using atomic force microscopy (AFM) under controlled humidity, where single pollen grains were attached to tipless AFM cantilevers. Pollen grains had an average adhesion of 10 +/- 3 nN with the surfaces, independent of surface type or relative humidity from 20% to 60%. van der Waals forces are the primary molecular attraction driving pollen adhesion to these surfaces. The results also indicate that ragweed pollen contacts the polymer surface via its exine surface spikes, and the total adhesion force scales with the number of contacts. The pollen surface spikes are strong, resisting fracture and compliance up to a load of 0.5 GPa.

Pollen discrimination and classification by Fourier transform infrared (FT-IR) microspectroscopy and machine learning

The discrimination and classification of allergy-relevant pollen was studied for the first time by mid-infrared Fourier transform infrared (FT-IR) microspectroscopy together with unsupervised and supervised multivariate statistical methods. Pollen samples of 11 different taxa were collected, whose outdoor air concentration during the flowering time is typically measured by aerobiological monitoring networks. Unsupervised hierarchical cluster analysis provided valuable information about the reproducibility of FT-IR spectra of the same taxon acquired either from one pollen grain in a 25 x 25 microm2 area or from a group of grains inside a 100 x 100 microm2 area. As regards the supervised learning method, best results were achieved using a K nearest neighbors classifier and the leave-one-out cross-validation procedure on the dataset composed of single pollen grain spectra (overall accuracy 84%). FT-IR microspectroscopy is therefore a reliable method for discrimination and classification of allergenic pollen. The limits of its practical application to the monitoring performed in the aerobiological stations were also discussed.

Cibirhiza spiculata (Apocynaceae), a remarkable new species from eastern Ethiopia

The new species Cibirhiza spiculata Thulin & Goyder, from Acacia-Commiphora bushland on sand in the Somali National Regional State (Ogaden) of Ethiopia, is described and illustrated. Its conservation status has been assessed. A molecular phylogenetic analysis confirms its placement in the tribe Fockeeae and indicates a position of the new species as sister to C. albersiana. The almost obsolete anther appendages, the outer corona with spreading lobes, as well as the pollinia with apparently single pollen grains, also support its placement in Cibirhiza. However, C. spiculata differs markedly from both previously known species of this genus (C. dhofarensis in Oman and Yemen and C. albersiana in Tanzania and Zambia) by its narrowly linear, subsessile leaves and by the inner corona lobes that are curved inwards over the gynostegium and each ending in a spinulose head.

High‐throughput Procedure for Single Pollen Grain Collection and Polymerase Chain Reaction in Plants

Single pollen grain polymerase chain reaction (PCR) has succeeded in several species, however only limited numbers of pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput (HT) procedure for collecting and detecting genetic variation in a large number of individual pollen grains by PCR is reported. The HT procedure involved the collection of individual pollen grains by a pair of special forceps and the lysis of pollen grains in a heated alkali/detergent solution followed by neutralization with a tris-ethylenediamine tetraacetic acid (TE) buffer. These resulting template solutions yielded PCR reactions involving the 5S ribosomal RNA intergenic spacers, randomly amplified polymorphic DNA, and simple sequence repeats markers. Using this procedure, one person with experience could collect and process up to 288 single pollen grain PCR reactions per day. The method worked well on sugarcane, corn, Miscanthus spp., snap bean, sorghum, and tomato. The ability to collect and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses and mapping studies in an easily controllable environment with a considerable cost reduction. The method will also significantly benefit studies in species that are difficult subjects for classical genetic research.

Development of electron spin resonance radical immunoassay for measurement of airborne orchard grass (Dactylis glomerata) pollen antigens

We have developed a highly sensitive method for the measurement of airborne orchard grass (Dactylis glomerata: Dac g) pollen antigens using an electron spin resonance (ESR) radical immunoassay. In this immunoassay, the lowest detectable level of Dac g antigen in a sample is 0.1 arbitrary unit; the amount of Dac g antigen in single pollen grains was found to be as 1.84 units. Thus, Dac g antigens can be detected in amounts of 1/20th of that contained in the grain. This immunoassay enables early detection of grass pollen antigens. Such information may be useful for patients with grass pollinosis, especially for those who show symptoms when only low levels of the pollen antigens are present in air. In this study, minor amounts of Dac g antigen (cross-reactive antigens) were detected in late March, after which the levels gradually increased. The levels were detected to be 10 units/m3 until the middle of May and then increased after blooming of orchard grass. High levels were maintained until the middle of June. Some patients who suffer from grass pollinosis show symptoms in late April and early May, when the airborne Dac g antigen levels were found to be 5–10 units/m3.

Pollen presentation, pollen-ovule ratios, and other reproductive traits in Mediterranean Legumes (Fam. Fabaceae - Subfam. Faboideae)

We studied the allocation of reproductive resources in 32 Mediterranean legume species. Correlations between main parameters were analysed at interspecific level. For each species we described the type of pollen presentation and examined its systematic significance. The high P/O values observed reflect low pollen transfer efficiency, typical of xenogamous species. Lowest P/Os were found in species with brush or explosive tripping mechanism, while highest ones were associated to primary pollen presentation. No trade-off was observed between P/O and the volume of a single pollen grain, nor between pollen number and pollen grain volume. Perennials showed higher P/Os compared to annuals, in accordance with the assumption that annual species are often self-compatible while most perennials are outcrossers. Fruit and seed set, evaluated in eight species under different experimental conditions, showed a close dependence on insects, and the existence of self-compatibility in three species (Colutea arborescens, Coronilla emerus, Hedysarum coronarium). In Colutea arborescens and Coronilla emerus pollen-pistil interaction and pre-zygotic barriers to selfing were investigated through stigma receptivity tests and in-vivo pollen germination trials. Results demonstrated the importance of the stigmatic cuticle in reducing levels of autogamy. Productivity tests confirmed the utility of P/O ratio as indirect measure of species' breeding system.

The determination of multiple microsatellite genotypes and DNA sequences from a single pollen grain

AbstractPollen plays important roles in the reproduction and gene flow of flowering plants, and its haploid DNA sequence provides useful information for studies of plant evolution and genealogy. We describe a new method for multiple microsatellite genotyping and DNA sequencing from a single pollen grain. The haploid DNA was extracted from a single pollen grain by using a simple DNA extraction method, and multiple microsatellite genotypes and DNA sequences of multiple chloroplast loci were determined. Using nine pairs of microsatellite primers, more than 90% of genotypes were successfully determined, and 71% and 100% of DNA sequences were determined at two chloroplast DNA loci, the trnL intron region and the trnL/trnF intergenic spacer region, respectively. This simple method of genetic analysis for a single pollen grain will facilitate detailed study of pollination, evolution and genealogy.

Vibrational Imaging of a Single Pollen Grain by Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy

Abstract Ultrabroadband coherent anti-Stokes Raman scattering (CARS) microspectroscopy using supercontinuum has been applied to investigate a single pollen grain at a molecular level. The CARS images due to carotenoid derivative and lipid have been successfully obtained with different vibrational contrast.

Millenial-scale climatic and vegetation changes in a northern Cerrado (Northeast, Brazil) since the Last Glacial Maximum

In the Southern Hemisphere, lacustrine sediments started to be deposited with the beginning of the deglaciation at ca 19,000 cal yr BP. At this time the region of Lake Caço was dominated by sparse and shrubby vegetation with dominance of steppic grasses in a poor sandy soil. The landscape did not present any ecological characteristics of a modern Cerrado. However single pollen grains of two Cerrado indicators, Byrsonima and Mimosa, suggest that some Cerrado species were able to survive under the prevailing arid climate, probably as small shrubs. After 15,500 cal yr BP, a sudden increase in the moisture rates is evidenced with the progressive expansion of rainforest showing successive dominance of various associations of taxa. The development of the forest stopped abruptly at the end of the Pleistocene between 12,800 and 11,000 cal yr BP, as attested by strong fires and the expansion of Poaceae. In the early Holocene an open landscape with a relatively high level of water in the lake preceded the progressive expansion of Cerrado species towards a denser forested landscape; fires are recorded from then on, resulting in the physiognomy of the Cerrado we know today. Late Pleistocene paleoenvironmental records from northern Brazil reflect the interplay between insolation forcing of two hemispheres with the local components represented by the interannual shift of the Inter Tropical Convergence Zone and the influence of seasonal equatorwards polar air incursions.

The tribal position of Fockea and Cibirhiza (Apocynaceae: Asclepiadoideae): evidence from pollinium structure and cpDNA sequence data

The pollinium morphology of the two members of the Asclepiadoideae, tribe Fockeeae, Fockea Endl. and Cibirhiza Bruyns, has been studied in detail and compared with that of eight genera of Marsdenieae, the tribe in which Fockea and Cibirhiza were previously accommodated and thus their putative closest relatives, as well as nine genera of Asclepiadeae. Both Fockea and Cibirhiza have several morphological characteristics in common, the most important of which is the absence of well-developed caudicula, which distinguishes them from all other genera of Asclepiadoideae known. The pollinium structure of these two genera, however, differs significantly. Whereas the pollinium of Cibirhiza consists of single pollen grains and is covered by a pollinium wall, as is typical for other Asclepiadoideae, the pollinium of Fockea consists of tetrads and is not covered by a pollinium wall, a condition otherwise typical of Secamonoideae. Fockea, however, has only two pollinia per anther, as does Cibirhiza and all other Asclepiadoideae, whereas the Secamonoideae have four pollinia per anther. Sequence data from two intergenic spacers, trnT-L and trnL-F and the trnL intron of cpDNA was analyzed. The ingroup included three species of Fockea and one species of Cibirhiza. The outgroup taxa consisted of three representatives each of Periplocoideae, and Secamonoideae and 24 species of Asclepiadoideae, including representatives of all tribes, of which eight genera belong to Marsdenieae, as outgroups. The results of the DNA analysis provide strong support for Fockeeae as a monophyletic tribe, distinct from Marsdenieae and, to the rest of the Asclepiadoideae. With the exception of pollen data, all morphological and molecular evidence clearly support recognition of the tribe Fockeeae. The occurrence of two such significantly different types of pollinia structure – characters elsewhere in the family used to distinguish subfamilies – within the small tribe Fockeeae was unexpected, and can perhaps best be understood as yet another attestment to the basal position of the Fockeeae in the nascence of the Asclepiadoideae.

Long-term history of land-use and vegetation at Ire, an agriculturally marginal area in Blekinge, south Sweden

Pollen records from a small lake and a small peatland at Ire in northern Blekinge, Sweden, reveal that until A.D. 300 there was a mixed deciduous type of woodland dominated by Quercus, Tilia and Corylus, with Betula, Pinus, Populus, Fraxinus, Ulmus and Acer as important constituents. The first, but weak, signs of human influence on the vegetation are detected around 2300 B.C. At this time, the area was probably used for woodland grazing. The regional expansion of Fagus occurred around 100 B.C.-A.D. 200, and later, at about A.D. 500, woods dominated by Fagus were common in the area. Around A.D. 600-700 an isolated farmstead may have been established in the area, as single pollen grains of Secale (rye) were found. This farmstead may represent the first permanent settlement in the area. A more widespread opening of the vegetation occurred around A.D. 1000, probably as an effect of a more pronounced use of the area, and an expansion of settlements in the region. The openness of the area seems to have peaked around A.D. 1400-1800, and during this period the vegetation was highly fragmented with small stands of woodland, intensively grazed pastures, and arable fields where Secale and Triticum were mainly cultivated. Around A.D. 1600 Fagus pollen percentages sharply decrease, most probably caused by a deliberate and selective felling of beech trees for the production of potash, which consumed enormous amounts of wood. Picea seems to have been established around A.D. 1600, but it did not become a regional dominant until the first part of the 20th century, when land use decreased and it became favoured by forestry. (Less)

Double fertilization in maize: the two male gametes from a pollen grain have the ability to fuse with egg cells.

In flowering plants, two male gametes from a single pollen grain fuse with two female gametes, the egg and central cells, to form the embryo and endosperm, respectively. The question then arises whether the two male gametes fuse randomly with the egg and central cells. We investigated this question using two nearly isogenic maize lines with supernumerary B chromosomes (TB10L18) or without (r-tester). B chromosomes regularly undergo non-disjunction at the second pollen mitosis, producing one sperm cell with zero B chromosomes and one with two. We first confirmed earlier studies showing an excess of transmission of the B chromosomes to the embryo rather than to the endosperm. We then tested the possibility of a directed fertilization. For TB10L18 pollen, we could demonstrate the existence of a size dimorphism between the two sperm cells, correlated to the content in B chromosomes, as detected by fluorescence in situ hybridization (FISH). However, no directed fusion of B chromosome containing sperm to egg cells could be detected when using in vitro fertilization. The absence of directed fusion in vitro could also be demonstrated for control lines. We conclude that both male gametes have the capacity to fuse with the egg cell in maize, although sexual reproduction results in a preferential transmission of supernumerary B chromosomes.

Pollen transfer patterns in a distylous plant with overlapping pollen‐size distributions

In this study we quantify the relative rates and amounts of between‐morph (legitimate) cross‐pollination, within‐morph (illegitimate) cross‐pollination and self‐pollination in a natural population of distylous Gaertnera vaginata (Rubiaceae) endemic to the oceanic island of La Réunion in the Indian Ocean. Since pollen size distributions of each morph overlap we develop a method, based on chi‐squared comparisons of observed to expected pollen size distributions, to quantify pollen transfer proficiencies. This method, unlike previous methods, does not assume an equal chance of pollen transfer for pollen grains in the region of overlap. The probability that a single pollen grain from a short‐styled plant undergoes legitimate pollen transfer to long‐styled flowers (4.19×10−2) was more than twice that for illegitimate transfer to other short‐styled plants (1.56×10−2). In contrast, the probability that a single pollen grain from a long‐styled flower undergoes legitimate pollen transfer to short‐styled flowers (1.65×10−2) was no greater than that of illegitimate pollen transfer to other long‐styled plants (2.15×10−2). The transfer proficiencies of individual pollen grains are thus highly asymmetric. However, because long‐styled flowers produce significantly more pollen, the ratio of legitimate to illegitimate pollen on the stigmas of short‐styled flowers (1.85) markedly exceeded that for long‐styled flowers (1.11). Short‐styled flowers incurred significantly higher levels of self‐pollination than long‐styled plants, emphasizing the importance for self‐pollination to be removed before comparing stigmatic pollen loads. The method we propose could be applied to other distylous species with pollen size overlap between morphs and thus allow for a more widespread study of the pollination ecology of heterostylous species.

Morphological and ultrastructural diversity of orbicules in relation to evolutionary tendencies in apocynaceae s.L.

Minute granules of sporopollenin, called orbicules, can be observed on the innermost tangential and/or radial walls of secretory tapetum cells. Orbicules were investigated in 62 species (50 genera) of Apocynaceae s.l. using light microscopy, scanning electron microscopy and transmission electron microscopy. Orbicules were found in 43 species (34 genera) distributed amongst the subfamilies Rauvolfioideae, Apocynoideae, Periplocoideae, and in the genus Riocreuxia (Asclepiadoideae). Absence of orbicules is apparent in Secamonoideae and Asclepiadoideae (except Riocreuxia). The orbicule types described are based on observed morphological and ultrastructural variation. Of the six orbicule types previously described, Type I and Type II orbicules are lacking. In the majority of species, Type III orbicules were recorded in addition to Types IV, V and VI. In this study we suggest that embedded Type VI orbicules are more derived. A correlation between orbicule typology and evolutionary tendencies in Apocynaceae s.l. palynology was found. A trend was observed from the presence of Type III orbicules in the majority of species belonging to the basal group of genera characterized by colporate to porate single pollen grains, or 3-6-porate tetrads, towards the more derived embedded Type VI orbicules in the more advanced Periplocoideae genera with multiporate tetrads or pollinia. Orbicule data have proven not to be useful for evaluating tribal delimitation within the Apocynaceae s.l. contrary to the Rubiaceae and Loganiaceae s.l.

Immunologic determination of the major allergen, Cry j 1, in Cryptomeria japonica pollen of 117 clones in Toyama prefecture: Some implications for further forestry research in pollinosis prevention

ABSTRACT Background A number of recent studies have clarified the characteristics of major allergens of Cryptomeria japonica, such as the locality, molecular weights of Cry j 1 and Cry j 2 and cross-reactivity with some other species of pollen. However, in order to plant C. japonica forests with low allergen levels, there is still a shortage of fundamental data for forest breeding. Methods To obtain the some fundamental data concerning Cry j 1, three items were investigated: (i) variations in weight and Cry j 1 content per single pollen grain among 117 clones; (ii) environmental factors affecting Cry j 1 content; and (iii) genotype variations of Cry j1. Results Although the variation in weight per single pollen grain among 117 clones was relatively small (mean (± SD) 10.2 ± 0.1 ng), the Cry j 1 content was found to vary widely from 0.26 to 7.54 pg. The expression level of Cry j 1 was higher in samples collected from lower altitudes. Using western blotting, it was shown that there were two Cry j 1 genotype variations. Conclusions From these results, we have concluded that immunologic determination of Cry j 1 by individual pollen grain levels provides useful data, especially for forest breeding to prevent pollinosis.

Rejection of S-heteroallelic pollen by a dual-specific s-RNase in Solanum chacoense predicts a multimeric SI pollen component.

S-heteroallelic pollen (HAP) grains are usually diploid and contain two different S-alleles. Curiously, HAP produced by tetraploids derived from self-incompatible diploids are typically self-compatible. The two different hypotheses previously advanced to explain the compatibility of HAP are the lack of pollen-S expression and the "competition effect" between two pollen-S gene products expressed in a single pollen grain. To distinguish between these two possibilities, we used a previously described dual-specific S(11/13)-RNase, termed HVapb-RNase, which can reject two phenotypically distinct pollen (P(11) and P(13)). Since the HVapb-RNase does not distinguish between the two pollen types (it recognizes both), P(11)P(13) HAP should be incompatible with the HVapb-RNase in spite of the competition effect. We show here that P(11)P(13) HAP is accepted by S(11)S(13) styles, but is rejected by the S(11/13)-RNase, which demonstrates that the pollen-S genes must be expressed in HAP. A model involving tetrameric pollen-S is proposed to explain both the compatibility of P(11)P(13) HAP on S(11)S(13)-containing styles and the incompatibility of P(11)P(13) HAP on styles containing the HVapb-RNase.

The cytoskeleton and polarization during pollen development in Carex blanda (Cyperaceae)

Patterns of cytoskeletal organization during distinct polarizations that characterize pollen development in the sedge Carex blanda (Cyperaceae) were studied by correlated methods of immunohistochemistry and confocal and transmission electron microscopy. As is typical of the family Cyperaceae, Carex produces a unique pollen type known as a pseudomonad in which all four microspores of the tetrad are enclosed within the wall of a single pollen grain. Only one member of the tetrad is functional and the other three abort. The pseudomonads are precisely oriented in the locule with the functional microspore in the wide abaxial portion of the wedge-shaped cytoplasm adjacent to the tapetum, and the degenerative microspores are packed tightly in the pointed adaxial portion. A unique sequence of post-meiotic developmental events reflects both intracellular and intercellular polarity. Development includes: (1) random placement of tetrad nuclei in the coenocytic sporocyte after meiosis, (2) interrupted cytokinesis resulting in a tetrad of nuclei that migrates as a unit into the narrow adaxial tip, (3) completion of unequal cytokinesis and centering of the functional nucleus in the wide abaxial portion of the microsporocyte via a radial array of microtubules and microfilaments, (4) unequal mitosis resulting in a small generative cell at the proximal surface of the functional microspore (adjacent to the abortive microspores), and (5) recentering of the vegetative nucleus in the abaxial cytoplasm via a radial cytoskeletal array.

Technical advance: single pollen typing combined with laser-mediated manipulation.

We combined single pollen typing with laser-mediated manipulation. After drilling a hole in the wall of a pollen grain from a dioecious plant (Silene latifolia) with a UV-laser microbeam, the single pollen grain was recovered directly in the cap of a PCR tube, using a non-contact method called laser pressure catapulting. The entire genome of the single pollen grain was then amplified with improved primer-extension-preamplification PCR (I-PEP PCR). Nested PCR with sequence tagged site (STS)-specific primers was used to analyze several loci in the haploid genome. The single copy gene MROS1 was detected in most of the single pollen grains analyzed. Bgl10, which is localized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using inverse PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The single pollen analysis using nested PCR showed that MROS3A and MROS3B are derived from different loci that are not located on the X chromosome. Single pollen typing not only reveals sex chromosome-linkage within the haploid genome, but can also discriminate between alleles and different loci. This method should also be useful for measuring recombination frequencies without genetic crossover analysis.