Changes in cytoplasmic carbohydrate content during Helleborus pollen presentation

Pollen grains of Helleborus foetidus and H. bocconei were exposed to low temperature treatments to simulate the natural events in pollen presentation of these two winter flowering species and to analyze the pollen carbohydrate content (glucose, fructose, sucrose and starch). In both species, cytoplasmic polysaccharides, monosaccharides and sucrose were found, while only Helleborus foetidus contained starch. Polysaccharide, sucrose and monosaccharide content varied as low temperature exposure time varied, a decrease in temperature decreases polysaccharide content and increases sucrose and monosaccharides. The relative quantities of the various types of carbohydrates were not affected by variations in the naturally occurring thermal cycles. Treatments did not greatly affect pollen viability. Although the occurrence of carbohydrates in pollen is known since many years, their function is still unclear. The findings of this research suggest a role of cytoplasmic pollen carbohydrates in resistance to low temperature exposure. The inter-conversion of carbohydrate type may be an adaptation for sustaining viability during pollen presentation that is particularly important for a winter flowering species such as Helleborus foetidus and H. bocconei .     

Classification and counting of fluorescent pollen using an image analysis system

Pollen viability is commonly assessed by fluorochromatic reaction (FCR) because of the high correlation between positive fluorescence of the pollen grains and their ability to germinate. One of the advantages of this method is its simplicity. An experiment to test FCR analysis for reproducibility, however, showed that results are affected by subjectivity. There is little consistency between analysts, and assessment by the same analyst may differ for the same pollen sample image examined at different times. These problems were solved by a computerized image analysis system that provides a method for classifying positive and negative fluorescent pollen and automatic counting of the grains in each class. The computerized image analysis system does not change the biochemistry of the FCR test, but avoids some experimental errors owing to the subjectivity of the analyst. Microscope images of the pollen after FCR were digitized and later analyzed by specially designed software, "Plant Meter." This software deletes the dark background of the image to isolate the grains, and subsequently counts positive and negative fluorescent pollen grains. An experiment was carried out to validate software output and it showed reliable results. Moreover, the software is user friendly and very little training is necessary for analysts to achieve reliable results.     

Fluorescein diacetate used as a vital stain for labeling living pollen tubes

Snapdragon and tobacco pollen treated with fluorescein diacetate (FDA, 10 μg/ml) for 40 min could germinate and grew well in FDA-free medium. The pollen tubes showed bright fluorescence of the protoplasm when they were living. Thus the FDA method can be used, in addition to its previous usage for viability test of cells, also for vital staining of pollen tubes, and may be valuable to the study of other living cells as well.     

Isolation and purification of generative cells from fresh pollen of Vicia faba L.

A technique named two-step osmotic shock was developed for isolating generative cells (GCs): pollen grains were incubated in 20% sucrose solution and shocked by adding water. This caused the pollen grains to burst and release their contents including GCs. By subsequent filtration and centrifugation the isolated GCs were purified. The viability of GCs before and after purification was confirmed by fluorescein diacetate (FDA) test. This procedure offers a rapid and effective means to obtain living GCs in quantities.Abbreviations GC Generative cell - FDA Fluorescein diacetate     

Light and electron microscopies reveal unknown details of the pollen grain structure and physiology from Brazilian Cerrado species

Pollen grains have a relatively simple structure and microscopic size, with two or three cells surrounded by the protective sporoderm at maturity. The viability and efficiency of pollen transport from anther to stigma depends on pollen physiological properties, especially the relative water content of the vegetative cell. Pollen transport is a crucial fate for most angiosperms that depends on biotic pollinators and studies focusing on understanding the morpho-physiological properties of pollen grains are still scarce, especially to tropical open physiognomies as the Brazilian Cerrado. Therefore, we investigate some structural and physiological aspects of pollen grains from six native species naturally growing in one Cerrado area: Campomanesia pubescens (Myrtaceae), Caryocar brasiliense (Caryocaraceae), Erythroxylum campestre (Erythroxylaceae), Lippia lupulina (Verbenaceae), Pyrostegia venusta (Bignoniaceae), and Xylopia aromatica (Annonaceae). We selected dehiscent anthers and mature pollen grains to analyze (1) the anther wall and pollen microstructure, (2) the pollen water status at the time of anther dehiscence, and (3) the pollen chemical compounds. In all analyzed species, the anther and pollen developed in a successfully way, and except for Caryocar brasiliense, all species were able to emit pollen tubes in the germination tests. As expected for a dry and open environment, most species dispersed their pollen grains in a partially dehydrated form, as indicated by our harmomegathy experiment. As indicated by our study, the pollen ability in preventing dissection, maintaining its viability in a dry and hot environment during its transport from anther to stigma, may be related to the sporoderm apertures and to the reserve compounds, mainly carbohydrates in the form of hydrolysable starch grains.

     

The effect of heat stress on tomato pollen characteristics is associated with changes in carbohydrate concentration in the developing anthers

Continuous exposure of tomato 'Trust' to high temperatures (day/night temperatures of 32/26 degrees C) markedly reduced the number of pollen grains per flower and decreased viability. The effect of heat stress on pollen viability was associated with alterations in carbohydrate metabolism in various parts of the anther during its development. Under control, favourable temperature conditions (28/22 degrees C), starch accumulated in the pollen grains, where it reached a maximum value 3 d before anthesis; it then diminished towards anthesis. During anther development, the concentration of total soluble sugars gradually increased in the anther walls and in the pollen grains (but not in the locular fluid), reaching a maximum at anthesis. Continuous exposure of the plants to high temperatures (32/26 degrees C) prevented the transient increase in starch concentration and led to decreases in the concentrations of soluble sugars in the anther walls and the pollen grains. In the locular fluid, however, a higher soluble sugar concentration was detected under the high-temperature regime throughout anther development. These results suggest that a major effect of heat stress on pollen development is a decrease in starch concentration 3 d before anthesis, which results in a decreased sugar concentration in the mature pollen grains. These events possibly contribute to the decreased pollen viability in tomato.     

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     

Effects of High Temperature Stress on Brassica Pollen: Viability, Germination and Ability to Set Fruits and Seeds

Pollen grains of Brassica juncea (L) Czern. were exposed tohigh temperature (45, 60 and 75°C, for 4–24 h) andtheir viability [using the fluorochromatic reaction (FCR) test],germination, and ability to set fruits and seeds were investigated.High temperature up to 60°C did not affect pollen viability;these pollen samples germinated on the stigma and produced fruitsand seeds. There was no correlation between viability and seedset in pollen samples exposed to 75°C; they showed onlyabout 25% reduction in viability but failed to germinate onthe stigma and, consequently, to set seeds. However, these pollensamples induced parthenocarpy. A clear relationship betweenviability and seed-set was observed when the extent of retentionof fluorescence in pollen over a 2-h period was used to assessviability. The fluorescence was retained even after 2 h in pollensamples which induced seed-set, while in those which failedto induce seed-set the fluorescence was lost in about 60 mindue to the leakage of fluorescein. Thus, in stressed pollen,the extent of retention of fluorescence may give a better indicationof pollen function than initial fluorescence, as is being doneroutinely in FCR test. Brassica, White Sim, carnation, Petal senescence, ultrastructure, ethylene, climacteric vacuoles, membranes, wall lysis     

Pollen starch reserves in tomato relatives: Ecophysiological implications

The presence or absence of starch in microspore development and in pollen grains was recorded in eleven wild tomato species (Solanum sect. Lycopersicon) and two close relatives (S. lycopersicoides and S. sitiens). In all the species starch started to accumulate in the early microspore bicellular stage and continued until the cytoplasm was filled. At flower anthesis, pollen grains were mostly starchless in the wild tomatoes, except in S. pennellii, which had starchy pollen. Starchy pollen is also present in the two related species. The latter two species had larger pollen grains and grow in drier environments than the other species. The heterogeneity of pollen starch content among all these species, supposed to have the same pollination mechanism, is a new finding supporting the idea that starch content and pollination mechanism do not necessarily influence each other. The presence of starchy pollen in the self‐incompatible species, which grow in the driest environments, raises questions regarding the relationship between carbohydrates content and pollen survival.     

Effect of Water Loss on Germination Ability of Maize (Zea mays L.) Pollen

The correlation between water content and viability of maizepollen grains was studied on the basis of the germination abilityof pollen from a single cross hybrid. There was found to beclose correlation between viability of the grains and theirtolerance to desiccation. Most of the pollen grains in the hybridexamined survived a reduction by almost 50 per cent of the originalwater content without loss of normal function. With water lossgreater than this, less vigorous pollen grains died or losttheir ability to form pollen tubes. Consequently, when pollinationwas carried out using pollen with a water content reduced bymore than 50 per cent, only the most tolerant pollen grainssurvived to take part in the competition which precedes fertilization.Dry pollen grains required a longer period to establish adhesionto the stigma surface and to initiate pollen tubes than pollengrains with higher water content, but otherwise their behaviourwas normal. If more than 80 per cent of the original water contentwas lost, disturbances occurred in the physiology of the grainssurviving the treatment. This was exhibited as death or a reductionin rate of pollen tube growth. Drying of pollen by an amount which does not irreversibly damagethe more tolerant grains could possibly be used as a means ofpollen selection. Zea mays L., maize, pollen viability, pollen treatment, dehydration, pollen tube     

Factors affecting in vitro maturation of isolated maize microspores

Summary An in vitro method to simulate pollen development was developed in maize (Zea mays L.). Microspores at the late uninucleate to early binucleate stage were isolated and cultured under various conditions. Cell viability, starch content and the formation of the three nuclei as found in normal mature pollen were monitored during the course of the culture. Media composition was modified in order to promote starch accumulation and frequency of mitosis, while maintaining the viability of the microspores. Under the best conditions, up to 12% of the microspores matured in vitro into trinucleate, starch-filled viable pollen grains which were unable to germinate or produce seeds. At different stages during in vitro maturation, proteins patterns were analyzed and compared with their in vivo equivalent and the patterns were only partially similar.     

Introduction of impermeable molecules into pollen grains by electroporation

Summary Electroporation was used to introduce plasma membrane impermeable molecules into the cytoplasm of pollen grains ofLilium longiflorum. Ungerminated pollen grains were exposed to the fluorescent dye quin2 or FITC-labelled dextrans and electroporated with exponentially decaying voltage pulses of 250 to 2000 V/cm and time constants of 0.01 to 10 s. The number of electroporated pollen grains increased with the strength and duration of the voltage pulses, and with the osmolarity of the external medium. Optimal results were obtained with pulses of 1000 V/cm and 10 s time constant, and with 900 mM mannitol in the electroporation buffer. The size of the pores produced in the plasma membrane by electroporation allowed uptake of 40 kDa dextran but not 70 kDa dextran. The rate of germination of pollen grains was low immediately after electroporation, but increased with time in pollen growth medium. The conditions of electroporation reported here may be used to load genetic material into pollen grains for the production of transgenic plants.Abbreviations PGM pollen growth medium - FDA fluorescein diacetate - FITC fluorescein isothiocyanate     

The legwd mutant uncovers the role of starch phosphorylation in pollen development and germination in tomato

Starches extracted from most plant species are phosphorylated. α-Glucan water dikinase (GWD) is a key enzyme that controls the phosphate content of starch. In the absence of its activity starch degradation is impaired, leading to a starch excess phenotype in Arabidopsis and in potato leaves, and to reduced cold sweetening in potato tubers. Here, we characterized a transposon insertion ( legwd::Ds ) in the tomato GWD ( LeGWD ) gene that caused male gametophytic lethality. The mutant pollen had a starch excess phenotype that was associated with a reduction in pollen germination. SEM and TEM analyses indicated mild shrinking of the pollen grains and the accumulation of large starch granules inside the plastids. The level of soluble sugars was reduced by 1.8-fold in mutant pollen grains. Overall, the transmission of the mutant allele was only 0.4% in the male, whereas it was normal in the female. Additional mutant alleles, obtained through transposon excision, showed the same phenotypes as legwd::Ds . Moreover, pollen germination could be restored, and the starch excess phenotype could be abolished in lines expressing the potato GWD homolog ( StGWD ) under a pollen-specific promoter. In these lines, where fertility was restored, homozygous plants for legwd::Ds were isolated, and showed the starch excess phenotype in the leaves. Overall, our results demonstrate the importance of starch phosphorylation and breakdown for pollen germination, and open up the prospect for analyzing the role of starch metabolism in leaves and fruits.     

A method to estimate pollen viability from pollen size variation

The mean diameter of viable pollen grains is approximately 13 μm greater than the mean diameter of inviable grains in Mimulus guttatus. We show that this difference is large enough to be detected by particle counters and that these machines can be used to obtain a rapid estimate of pollen viability. While requiring a separate calibration, a size-based statistic is also strongly correlated with pollen viability in Collinsia verna. These results suggest that statistics derived from the size distribution of pollen grains may provide an alternative to more labor-intensive methods for estimating pollen viability, particularly in cases where inviability results from inbreeding depression or hybrid failure.     

Flow Cytometric Characteristics of Sperm Cells Isolated from Pollen of Zea mays L

Sperm cells have been isolated from pollen of maize (Zea mays L.) and purified with Percoll density centrifugation. Their flow cytometric characteristics were determined on a FACScan flow cytometer with the fluorescent dyes, fluorescein diacetate and propidium iodide. Freshly isolated sperm cells appeared as a dot cluster on the forward scatter and side scatter dot plot. This dot cluster contained 85 to 95% of the 10 thousand counts collected. More than 98% of cells from the cluster were fluorescein diacetate positive, with no propidium iodide positivity, indicating high cell viability. After 5 hours in 15% (w/v) sucrose at room temperature (23°C), scattering properties, cell number, and percentage of fluorescein diacetate-positive cells remained the same. In contrast, Brewbaker and Kwack salts in 15% sucrose resulted in the emergence of a new cell population, as well as a decrease in cell number at 5 hours. Further investigations with individual components of the Brewbaker and Kwack salts showed that calcium was mainly responsible for the deleterious effects. These results demonstrate the utility of flow cytometry as a tool to determine viability and to monitor morphological changes of plant sperm cells and to challenge current views on the ability of Brewbaker and Kwack salts to maintain viability of isolated sperm cells.     

Direct observations on generative cells isolated from pollen grains of Haemanthus katherinae baker

Generative cells from mature pollen grains of Haemanthus katherinae Baker (African blood lily) were isolated by means of a simple squash method and observed by differential interference contrast (DIC), fluorescence and polarizing microscopy. The isolated cells appeared structurally similar to those observed in vivo and gave no evidence of a typical cell wall. Their viability was confirmed using the fluorescein diacetate test. The cell shape changed rapidly as the sucrose concentration of the medium was varied. The squash method of isolating generative cells holds promise for the direct and experimental study of these cells, especially in the living state.Abbreviations FDA Fluorescein diacetate - PAS Periodic-acid-Schiff - DIC differential interference contrast - NA numerical aperture     

Viability and sunlight sensitivity of oak pollen and its implications for pollen-mediated gene flow

Pollen-mediated gene flow and the male reproductive success of wind-pollinated trees depend on the initial viability of the pollen and the changes that occur in its viability during transport in the atmosphere. The viability of Quercus robur pollen was determined before and during exposure to sunlight by in vitro germination and the fluorescein diacetate reaction (FCR) in 2002 and 2003, respectively. These experiments allowed us to calculate initial pollen viability and pollen sensitivity to sunlight. The germination test revealed a lower initial pollen viability (25–65%) than the FCR (53–92%). Following 9.5 h of irradiation the viability was reduced to 75–100% as determined by the in vitro germination test or to 40–70% as determined by the FCR. The actual values of initial pollen viability and pollen sensitivity to sunlight were used to define a range of values for modelling pollen dispersal using the mesoscale meteorological model METRAS. The deposition patterns of viable pollen varied by as much as a factor of 14 by changing the viability parameters in the range of the observed values. This suggests significant differences in male reproductive success. Variations in initial pollen viability have stronger effects on the gene-flow pattern than do variations in pollen sensitivity to sunlight. In particular, pollen distribution throughout the local environment is shaped by the initial pollen viability, while pollen sensitivity to sunlight mainly influences long-distance pollen dispersal.This revised version was published online in February 2005 with corrections to an incorrect sentence in the Results section. Under Pollen Sensitivity it should read: decreased faster when assessed by the germination test than by the FCR.     

Effects of γ-ray treatment onCannabis saliva pollen viability

The viability and thein vitro germination capability of hemp pollen (cv. Carmagnola) were studied. Viability tests were based on the microscopic observation of the fluorescence of loaded fluorescein diacetate (FDA), while, for germinability tests, five different media were tested. The effects of irradiation with γ-rays on pollen viability and germination and on seed set were also studied, at three different irradiation doses (20, 60 and 100krad). The results show that in one of the media tested, about 85–90% of the pollen grains are viable and able to germinate in control samples, and that while viability measured by FDA test is not affected by increased γ-ray doses, the pollenin vitro germinability drops to about one-half of the controls at the maximum γ-ray dose employed, 100krad. Seed set of hemp plants pollinated with the irradiated pollen dropped to less than 1% of that of plants pollinated by untreated pollen for the higher dose used. The different media suitable forin vitro germination of hemp pollen, and the observed lack of correspondence between viability and germination capacity tests are discussed.     

Pollen carbohydrates and water content during development, presentation, and dispersal: a short review

Summary. Pollen accumulates starch reserves during development and the final stage of ripening. Before the anther opens, starch is totally or partially converted to pectins, glucose, fructose, sucrose, and to some unknown polysaccharides. Pollen is exposed to dispersing agents in an arrested developmental state which differs according to pollen water content. Pollen is classified as partially dehydrated or partially hydrated. The final water content may be reached before or after anther opening. Especially during exposure and dispersal, partially dehydrated pollen may interconvert soluble and insoluble reserves, modifying internal turgor pressure and hindering water loss or gain. Partially hydrated pollen is commonly devoid of mechanisms to conserve viability in time but has the advantage of quickly emitting pollen tubes on reaching the stigma. Correspondence and reprints: Department of Environmental Sciences, Siena University, 4 Via P.A. Mattioli, 53100 Siena, Italy.     

DNA uptake during electroporation of germinating pollen grains

Electroporation of germinating pollen grains of Nicotiana gossei (L.) Domin under a variety of conditions showed that DNA was taken up by the pollen without detrimental effects on the viability of the pollen. By optimizing both the field strength of the electroporation pulse and the DNA concentration in the electroporation medium up to 6% of the donor DNA can be taken up by the germinating pollen while maintaining a pollen viability of 90%. Field strengths as high as 9 kV/cm could be applied to germinating pollen grains without detrimental effects on viability. Southern hybridizations demonstrated that DNA encoding the marker enzyme β-glucuronidase (GUS) was incorporated into electroporated pollen. Germinating pollen, treated in this manner, is capable of producing 300–400 seeds per capsule of viable seed when applied to the stigmas of compatible flowers of N. gossei which has been emasculated 4 days earlier.