Interspecific size and number variation in pollen grains and seeds

An interspecific correlation between pollen grain size and seed size is demonstrated by means of the phylogenetic regression, which allows for phylogenetic bias. The correlation was not explained by plant size, mass of DNA per cell, style length or breeding system, although the first three of these factors all correlated with both pollen size and seed size. Two interpretations, involving pollen competition and flower size, are discussed. There is also an interspecific correlation between pollen grain number per flower and ovule number per flower. Some consequences of these correlations for the interpretation of pollen-ovule ratios and sex allocation strategies are considered.     

Micromanipulation and in vitro fertilization with single pollen grains of maize

Summary The manipulation of single pollen grains of maize was studied. The effects of delivering substances both locally to the grain wall, tube or tip by a microcapillary and directly into the pollen grain by microinjection, and single grain pollination were investigated. Germination was induced by adding small amounts of water locally to the grains with either a microcapillary or with a waterdelivering emulsion without any other ingredients in the medium. The grains were overlayered by mineral or silicone oil so that tube growth proceeded without the grains bursting. There was no apparent penetration of high-molecular-weight substances (FITC-dextran, ethidium bromide labelled DNA) into the living grain either before or after pollination. Neither could the penetration of these substances be detected in both dry, viable and hydrated grains, tubes and tube tips, with or without treatment with Triton X-100 and dimethyl sulfoxide. By microinjection, however, the delivery of high-molecular-weight substances into grains was possible. Such injected grains successfully pollinated stigmas of cultured ear segments. Pollination with pore-injected grains was most efficient (mean 26%). No difference in fertilization rates between mass pollination (mean 41%) and single grain pollination (mean 39%) could be found. A mean fertilization rate of 29% could be obtained after microinjection. Seedlings developed 3 weeks after being pollinated by means of the in vitro pollination and fertilization method.     

Emerging data on pollen tube growth and fertilization in flowering plants, 1990–1995

Summary Since its discovery at the end of the last century, double fertilization remains of central interest in plant reproductive biology research. Although the sequence of events leading to fertilization is well known from cytological studies, the underlying mechanisms remain to be elucidated. This now seems feasible by the diversification and refinement of recently developed technologies presented in this review. The progress made during the last five years in understanding pollen tube guidance, discharge into the embryo sac, and gametic fusion are described. Future directions are also discussed.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

The relationship between airborne pollen distribution and the frequency of specific pollen sensitization at two climatically different locations in Switzerland

Pollen data reported from the two monitoring stations of Locarno-Monti, on the southern slopes of the Alps, and Zurich on the northern slopes of the Alps, for the years 1989–1993 were compared with meteorological data from the corresponding locations and with the prevalence of pollinosis and frequency of specific pollen sensitization in patients with pollinosis. It was so discovered that the recorded quantity of allergenic pollen types (Alnus, Corylus, Betula, Fraxinus, Poaceae, Castaneae,Olea andArtemisia) south of the Alps is higher by a factor of 2.9 than that north of the Alps. These differences are due to the distinctly milder climate in the canton of Ticino (south of the Alps) showing higher annual mean temperatures, more sunshine duration and less precipitation days with higher amounts of precipitation. The allergological data show additionally that the summer pollinosis, caused byCastanea andOlea, is probably responsible for the differing prevalence of pollinosis at the two sites. Finally, we can summarize that in Switzerland pollen from Poaceae, Betulaceae and Oleacea are the most important for pollinosis.     

Storage of pollen grains of Crotalaria retusa in oils

Summary Attempts were made to store pollen grains of Crotalaria retusa L. in a mineral oil (paraffin oil) and two vegetable oils (soybean oil and olive oil). Under laboratory conditions pollen grains not stored in oil lost in vitro germinability within 15–30 days, while those stored in oils maintained some degree of germinability even after 60 days. Pollen samples stored in oils at –20° C did not show any decline in germinability or pollen tube vigour even after 6 months of storage. The results amply demonstrate the feasibility of using oils for short- and long-term pollen storage.     

Covalently bound wall proteins of pollen grains and pollen tubes grown in vitro and in styles after self- and cross-pollination in Lilium longiflorum

Summary A method was worked out using trifluoromethanesulfonic acid (TFMS) as a reagent to split the covalently bound proteins, which are NaCl insoluble, from pollen tube walls of Lilium longiflorum, leaving the peptide bonds essentially intact. After electrophoretic separation, comparisons were made among these proteins from pollen grains and pollen tubes grown in vitro and in styles after self- and cross-pollination. It was found that a) the patterns of covalently bound wall proteins were different between tubes grown in vitro and in vivo; b) fewer bands were found in covalently bound wall proteins than that in noncovalently bound proteins; c) the bands remained almost the same no matter whether the tubes had been cross pollinated or self pollinated, indicating that while the noncovalently bound proteins were involved in incompatibility as shown in the previous paper, the covalently bound proteins may only serve as a structural component, having little to do with incompatibility.     

Airborne pollen grains in Nsukka,Nigeria

A study of the airborne pollen grains in Nsukka, Nigeria, has been carried out at two different sampling heights (1.8?m and 15?m) from February 1993 to January 1994. Twenty‐six plant families (40 genera) were identified at the lower sampling height, whilst thirty‐eight families (58 genera) were identified at the height of 15?m. A total of nine and eighteen fern spore types were observed at 1.80?m and 15?m, respectively. The quantitative results indicate that the number of pollen observed at 15?m sampling height was statistically different (p<0.05) from that observed at the height of 1.80?m. The analysis of airborne pollen grains indicates three different periods: (1) dry season, (2) rainy season, and (3) late rainy season to early dry season/Harmattan. The highest pollen abundance was recorded during the late rainy season – early dry season/Harmattan followed by that of the dry season. The predominant pollen grains and fern spores trapped at both heights include Poaceae, Casuarina equisetifolia, Milicia excelsa, Elaeis guineensis, Celtis integrifolia, Alchornea cordifolia, Amaranthaceae/Chenopodiaceae, Combretaceae/Melastomataceae, Nephrolepis biserrata, Thelypteris totta, and Dryopteris spp.     

The number of cells in the pollen of Melastomataceae (Myrtales)

The mature pollen of Melastomataceae is not three-celled, as reported earlier, but two-celled. Two-celled mature pollen is characteristic of all Myrtales, excluding Thymelaeaceae.     

Growth and development of conifer pollen tubes

Conifer pollen tubes are an important but underused experimental system in plant biology. They represent a major evolutionary step in male gametophyte development as an intermediate form between the haustorial pollen tubes of cycads and Ginkgo and the structurally reduced and faster growing pollen tubes of flowering plants. Conifer pollen grains are available in large quantities, most can be stored for several years, and they grow very well in culture. The study of pollen tube growth and development furthers our understanding of conifer reproduction and contributes towards our ability to improve on their productivity. This review covers taxonomy and morphology to cell, developmental, and molecular biology. It explores recent advances in research on conifer pollen and pollen tubes in vivo, focusing on pollen wall structure, male gametophyte development within the pollen wall, pollination mechanisms, pollen tube growth and development, and programmed cell death. It also explores recent research in vitro, including the cellular mechanisms underlying pollen tube elongation, in vitro fertilization, genetic transformation and gene expression, and pine pollen tube proteomics. With the ongoing sequencing of the Pinus taeda genome in several labs, we expect the use of conifer pollen tubes as an experimental system to increase in the next decade.     

Endocytotic uptake of fluorescent dextrans by pollen tubes grown in vitro

Summary Pollen tubes grow by tip growth, with high levels of exocytosis at the apex. The commercial availability of FITC labelled -linked dextrans provides a source of biologically inert tracers for endocytotic activity in pollen tubes. Growing tubes ofNicotiana andTradescantia were transferred to media containing 1% FD-4 for varying period of time before washing in control media and observation in a fluorescence microscope. Fluorescent material appeared to enter the pollen tubes only at the tip region, and to accumulate in vacuoles, starting with smaller vacuoles near the tip and spreading to the main vacuolated part of the tube. Mature tubes, with callose plugs, were only labelled up to the first complete plug from the tip, younger tubes without plugs were labelled into the pollen grain vacuole. The fluorescent material within the pollen tubes was shown to represent uptake of intact high molecular weight dextran by the following criteria: (i) free FITC and low molecular weight dextrans could not be detected in any of the media or pollen tubes using thin layer chromatography and (ii) pollen tube growth rates were unaffected by the fluorescent dextran, but were severely inhibited by low levels of free FITC. It was concluded that the dextrans entered the tubes by endocytosis, possibly in the tip region, and were then transferred to the vacuole system of the pollen tube.Abbreviations FITC fluorescein isothiocyanate - FD fluorescent dextran     

Postmeiotic cytokinesis and pollen aperture number determination in eudicots: effect of the cleavage wall number

Summary.  In eudicot postmeiotic tetrads, apertures are usually joined in pairs in highly conserved areas. These appear to be located at the last points of contact persisting at the end of cytokinesis between the cytoplasm of the future microspores. In order to investigate the relationship between cytokinesis and aperture formation, aperture distribution within postmeiotic tetrads and the progression of meiosis were studied in Nicotiana tabacum cv. Ambalema. This variety (inbred line) produces about 85% tricolporate pollen and 15% tetracolporate pollen grains. In addition, about 7% of tetrads are composed of four equal-sized microspores and a supernumerary pseudomicrospore of small size and an equal proportion of tetrads exhibit unpaired apertures (these apertures are not joined in pairs within tetrads). Observation of cytokinesis indicates that both unpaired apertures and pseudomicrospores could result from the persistence of late communications between microsporocytes. Observations of tetrads indicate that an increase in the number of elements that are separated during cytokinesis is correlated with an increase in microspore aperture number. All data converge to support the hypothesis that aperture site determination is partly controlled by the number of walls formed to separate the different elements of the tetrad. Received May 22, 2002; accepted October 29, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Laboratoire de Ecologie, Systematique et Evolution, Batiment 362, Université Paris Sud, 91405 Orsay cedex, France.     

Viral particles in developing pollen grains of Olea europaea

Double-walled tubules containing rows of isodiametric virus particles were observed in developing pollen grains of Olea europaea L. cultivar Correggiolo. Sometimes the tubules are contained in another double-walled tubular structure or in a tubular endoplasmic reticulum cistern. The viruses are present in the cytoplasm from the microspore mother cell stage up to the microspore stage but just before the first haploid mitosis they are to be found only in the pores, inside the evaginations formed by the plasmalemma. During the last phase of pollen grain development, after the germinative pores are completed, the viruses disappear.Abbreviations ER endoplasmic reticulum     

The influence of relative humidity on the swelling of pollen grains in vitro

The volume of hydrated pollen grains of Petunia hybrida L. during swelling in germination medium increased three times. The volume of desiccated pollen grains increased only two times after transfer to the same medium. This difference in swelling ability is attributed to different rigidities of the pollen grain wall,ccaused by the different hydration states. The relationship between pollen grain swelling and germination metabolism with regard to relative humidity is discussed.Abbreviation RH relative humidity     

Induction of haploidy from pollen grains in angiosperms — the current status

Summary Since the successful induction of haploids from anthers cultured in vitro in 1964, a great deal of attention has been given to this problem by those interested in obtaining pure lines and mutants for crop improvement and biochemical genetics. In the last 16 years the anther culture technique has been refined and extended to over one hundred and fifty different species. More recently, isolated pollen culture — which is a refinement of the original anther culture technique — has also been developed. In this review we have made an effort to critically examine existing reports with the objective of analysing the effects of various factors — e.g. culture medium, the cultural conditions, and the effect of genotype and physiological state of the parent plant on pollen induction — and to speculate on the mechanism of action of different factors in order to throw some light on the process of haploid induction.     

The relationship between pollen grain size and progeny gender in dioecious Silene latifolia (Caryophyllaceae)

Summary Experiments and observations conducted during the past 90 years have provided conflicting evidence concerning the existence of a size difference between pollen grains containing an X chromosome (female-determining) and those containing a Y chromosome (maledetermining) in dioecious Silene latifolia. Were such a size difference to exist, this might explain, at least in part, the observation that X-bearing pollen tubes reach the ovary more quickly, on average, than Y-bearing pollen tubes. We tested for such a size difference by separating pollen collected from single anthers into three size classes: small, large, and random. Fruit set (number of pollinated flowers that set fruit) and seed set (number of seeds per capsule) did not differ for these three pollination treatments. Progeny sex ratios resulting from these three pollen size classes also did not differ significantly. Thus, pollen grain size is not affected by which of the two sex chromosomes is present. Our experiment is the first direct test of this relationship. Based on our results, size differences should not be invoked to explain competitive differences in male- and female-determining microgametophytes.     

Dose effect of UV-B irradiation on pollen tube growth and seed-specific promoter activities in irradiated pollen grains of Nicotiana plumbaginifolia

Low doses of UV-B irradiation applied to mature Nicotiana plumbaginifolia pollen grains stimulated pollen tube growth. The most pronounced effect was achieved after 1.5 min of irradiation. Using transgenic N. plumbaginifolia plants expressing the GFP reporter gene under the control of the seed-specific promoters USP (unknown seed protein) or LegB4 (legumin B4) genes, it was shown that these promoters are also inducible by UV-B irradiation of the pollen grains. The improvement of pollen viability and germination by UV-light is discussed with respect to effects on plant flowering and reproduction. Received: 10 November 1999 / Revision accepted: 14 February 2000     

In vitro chemotropism of pearl millet pollen tubes to stigma tissue: a response to glucose produced in the medium by tissue-bound invertase

Summary Water-homogenized stigma pellets of pearl millet and precipitates resulting from dialysis of their salt extracts were observed to: (1) chemotropically attract pearl millet pollen tubes on a sucrose-containing pollen germination and growth medium, (2) have acid invertase activity as assayed by the arsenomolybdate method, (3) hydrolyze sucrose in the pollen germination and growth medium to glucose as assayed by coupled glucose oxidation with Nitro Blue Tetrazolium, and (4) lose chemotropic and invertase activities upon heat treatment. The results indicate that the in vitro chemotropic attraction of pearl millet pollen tubes to water-homogenized stigma pellets is a response to glucose produced by homogenate-pellet-bound invertase hydrolyzing the sucrose present in the pollen germination and growth medium. Yeast and tomato invertases used as controls verified this conclusion. Water extracts of whole stigmas contained water-soluble acid invertase. The results are discussed in relation to the identification of possible in vivo chemotropic factors of pearl millet and other plants by in vitro assays.Abbreviations dH₂O Deionized, house-distilled water - NBT Nitro Blue Tetrazolium, NBT-medium - PGG medium, pollen germination and growth medium (10% sucrose, 1 mM H₃BO₃, and 1% agarose); - WHS pellet, water-homogenized stigma pellet On Specific Cooperative Agreement 58-6612-8-002 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

Occurrence of mono- or disaccharides and polysaccharide reserves in mature pollen grains

 Pollen from 13 species of gymnosperms and angiosperms was studied for soluble and insoluble carbohydrates at dispersal. Starch reserves stored during pollen development give rise to carbohydrates at maturity. Combinations of different types of carbohydrates in mature pollen may depend on the extent of starch hydrolysis. An inverse relationship was found between the extent of starch hydrolysis and sucrose content. If the starch was scarcely de-polymerized, the cytoplasm had very low levels of soluble sugars and none of the periodic acid-Schiff (PAS)-positive material as found in pollen not subject to high dehydration (Cucurbita pepo L., Zea mays L.). After total or partial starch hydrolysis, insoluble PAS-positive oligo/polysaccharides were found in the cytoplasm associated with much soluble sugar, and the pollen grains were dehydrated at dispersal as in Typha latifolia L., Chamaerops humilis L., Trachycarpus excelsa Wendl., and other specimens. Intermediate levels of starch and soluble sugars, together with cytoplasmic PAS-positive material, characterized species with dehydrated pollen such as Pinus halepensis Miller. Carbohydrates may be related to pollen longevity, which largely depends on the abundance of sucrose, which is known to protect membrane integrity. The relationship between PAS-positive material and pollen viability is unclear at present. Received: 30 July 1996 / Revision accepted: 18 December 1996     

A style-specific 120-kDa glycoprotein enters pollen tubes ofNicotiana alata in vivo

Pistils ofNicotiana alata (Link et Otto) contain an abundant, style-specific glycoprotein (120 kDa) that is rich in hydroxyproline and has both extensin-like and arabinogalactan-protein-like carbohydrate substituents. An antibody specific for the protein backbone of the glycoprotein was used to localise the glycoprotein in both unpollinated and pollinated pistils. The glycoprotein is evenly distributed in the extracellular matrix of the style transmitting tract of unpollinated pistils and, despite the presence of extensin-like carbohydrate substituents, is not associated with the walls of the transmitting tract cells. In pollinated pistils the 120-kDa glycoprotein is concentrated in the extracellular matrix adjacent to pollen tubes, and is also present in the cytoplasm and the cell walls of pollen tubes. Pollen tubes grown in vitro do not contain the 120-kDa glycoprotein unless it is added to the growth medium, suggesting that the 120kDa glycoprotein located in pistil-grown pollen tubes is derived from the extracellular matrix of the transmitting tract.     

Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum

The distribution of cellulose and callose in the walls of pollen tubes and grains of Nicotiana tabacum L. was examined by electron microscopy using gold-labelled cellobiohydrolase for cellulose and a (1,3)-β-D-glucan-specific monoclonal antibody for callose. These probes provided the first direct evidence that cellulose co-locates with callose in the inner, electron-lucent layer of the pollen-tube wall, while both polymers are absent from the outer, fibrillar layer. Neither cellulose nor callose are present in the wall at the pollen-tube tip or in cytoplasmic vesicles. Cellulose is first detected approximately 5–15 μm behind the growing tube tip, just before a visible inner wall layer commences, whereas callose is first observed in the inner wall layer approximately 30 μm behind the tip. Callose was present throughout transverse plugs, whereas cellulose was most abundant towards the outer regions of these plugs. This same distribution of cellulose and callose was also observed in pollen-tube walls of N. alata Link et Otto, Brassica campestris L. and Lilium longiflorum Thunb. In pollen grains of N. tabacum, cellulose is present in the intine layer of the wall throughout germination, but no callose is present. Callose appears in grains by 4 h after germination, increasing in amount over at least the first 18 h, and is located at the interface between the intine and the plasma membrane. This differential distribution of cellulose and callose in both pollen tubes and grains has implications for the nature of the β-glucan biosynthetic machinery. Received: 20 February 1988 / Accepted: 25 March 1998