Hidden Pitfalls of Using Onion Pollen in Molecular Research.

There is little information on the use of pollen in molecular research, despite the increased interest in genome editing by pollen-mediated transformation. This paper presents an essential toolbox of technical procedures and observations for molecular studies on onion (Allium cepa L.) pollen. PCR is a useful tool as an express method to evaluate editing results before pollination. A direct PCR protocol for pollen suspension has been adapted without needing DNA pre-extraction. We showed that the outer layer of lipids known as pollenkitt is a limiting factor for successful PCR on pollen. A simple pre-washing step of pollen suspension was able to eliminate the pollenkitt and enormously affect the PCR results. Additionally, our pollenkitt study helped us develop a simple and effective pollination method using wetted onion pollen grains. Classical manual pollination usually is conducted by intact pollen without wetting. Most existing methods of the editing system delivery into pollen are carried out in a wet medium with consequent drying before pollination, which adversely affects the viability of pollen. The optimal medium for wet pollination was 12% sucrose water solution. Our method of using wetted pollen grains for pollination might be very beneficial for pollen genetic manipulation.

Cytochemical and comparative transcriptome analyses elucidate the formation and ecological adaptation of three types of pollen coat in Zingiberaceae.

BACKGROUND: The pollen ornate surface of flowering plants has long fascinated and puzzled evolutionary biologists for their variety. Each pollen grain is contained within a pollen wall consisting of intine and exine, over which the lipoid pollen coat lies. The cytology and molecular biology of the development of the intine and exine components of the pollen wall are relatively well characterised. However, little is known about the pollen coat, which confers species specificity. We demonstrate three types of pollen coat in Zingiberaceae, a mucilage-like pollen coat and a gum-like pollen coat, along with a pollen coat more typical of angiosperms. The morphological differences between the three types of pollen coat and the related molecular mechanisms of their formation were studied using an integrative approach of cytology, RNA-seq and positive selection analysis. RESULTS: Contrary to the 'typical' pollen coat, in ginger species with a mucilage-like (Caulokaempferia coenobialis, Cco) or gum-like (Hornstedtia hainanensis, Hhn) pollen coat, anther locular fluid was still present at the bicellular pollen (BCP) stage of development. Nevertheless, there were marked differences between these species: there were much lower levels of anther locular fluid in Hhn at the BCP stage and it contained less polysaccharide, but more lipid, than the locular fluid of Cco. The set of specific highly-expressed (SHE) genes in Cco was enriched in the 'polysaccharide metabolic process' annotation term, while 'fatty acid degradation' and 'metabolism of terpenoids and polyketides' were significantly enriched in SHE-Hhn. CONCLUSIONS: Our cytological and comparative transcriptome analysis showed that different types of pollen coat depend on the residual amount and composition of anther locular fluid at the BCP stage. The genes involved in 'polysaccharide metabolism' and 'transport' in the development of a mucilage-like pollen coat and in 'lipid metabolism' and 'transport' in the development of a gum-like pollen coat probably evolved under positive selection in both cases. We suggest that the shift from a typical pollen coat to a gum-like or mucilage-like pollen coat in flowering plants is an adaptation to habitats with high humidity and scarcity of pollinators.

Optimal pollen stickiness to pollinators for maximizing paternal fitness: Increased number of recipient flowers or increased pollen deposition on recipient flowers?

A plant can sire more seeds by increasing the number of pollen recipient flowers or the amount of pollen deposited on recipient flowers. We theoretically analyzed how pollen stickiness contributes to paternal fitness through changing the pattern of pollen dispersal including both the number of recipient flowers and overall pollen deposition (the overall amount of pollen deposited on recipient flowers) in animal-pollinated plants. We developed a numerical model in which pollen stickiness to pollinators increases with production of expensive materials on pollen surfaces, and a high level of stickiness diminishes the proportions of pollen lost from a pollinator body during a flight and pollen deposited on a stigma during a visit. We found that the number of recipient flowers monotonically increased with increasing pollen stickiness allocation while overall pollen deposition was maximized at a certain amount of stickiness allocation. We demonstrated that evolutionarily stable pollen stickiness attained many recipient flowers at the expense of overall pollen deposition in most cases while it merely favored maximization of overall pollen deposition in all other cases. Sticky pollen evolved if pollinators were highly likely to drop pollen during flights and did not diffuse well. In this situation, the evolutionarily stable pattern of pollen dispersal was acquisition of many pollen recipient flowers rather than maximization of overall pollen deposition. Sticky pollen also evolved if additional sticking elements were moderately effective in increasing the force of adhesion to pollinators. Pollen stickiness has a significant effect on the pattern of pollen dispersal via the extent of pollen carryover, and our results suggest that plants maximize paternal fitness by giving pollen the optimal stickiness, which varies with pollinating partners.

Flower orientation in Gloriosa superba (Colchicaceae) promotes cross-pollination via butterfly wings.

BACKGROUND AND AIMS: Complex modifications of angiosperm flowers often function for precise pollen placement on pollinators and to promote cross-pollination. We explore the functional significance of the unusually elaborate morphology of Gloriosa superba flowers, which are divided into one hermaphrodite meranthium and five male meranthia (functional pollination units of a single flower). METHODS: We used controlled pollination experiments, floral measurements, pollen load analyses and visitor observations in four populations of G. superba in South Africa to determine the breeding system, mechanism of pollination and role of flower in the promotion of cross-pollination. KEY RESULTS: We established that G. superba is self-compatible, but reliant on pollinators for seed production. Butterflies, in particular the pierid Eronia cleodora, were the primary pollinators (>90 % of visitors). Butterflies brush against the anthers and stigma during nectar feeding and pollen is carried on their ventral wing surfaces. Butterfly scales were positively correlated with the number of pollen grains on stigmas. We demonstrate that the styles were orientated towards clearings in the vegetation and we confirm that the highest proportion of initial visits was to hermaphrodite meranthia pointing towards clearings. CONCLUSIONS: The flower morphology of G. superba results in effective pollen transfer on the wings of butterfly visitors. The style-bearing hermaphrodite meranthium of the flowers orientates towards open spaces in the vegetation, thus increasing the probability that butterflies land first on the hermaphrodite meranthium. This novel aspect of flower orientation is interpreted as a mechanism that promotes cross-pollination.

Characterization of Optical, Thermal, and Viscoelastic Properties of Pollenkitt in Angiosperm Pollen Using In-Line Digital Holographic Microscopy.

Pollen grains are remarkable material composites, with various organelles in their fragile interior protected by a strong shell made of sporopollenin. The outermost layer of angiosperm pollen grains contains a lipid-rich substance called pollenkitt, which is a natural bioadhesive that helps preserve structural integrity when the pollen grain is exposed to external environmental stresses. In addition, its viscous nature enables it to adhere to various floral and insect surfaces, facilitating the pollination process. To examine the physicochemical properties of aqueous pollenkitt droplets, we used in-line digital holographic microscopy to capture light scattering from individual pollenkitt particles. Comparison of pollenkitt holograms to those modeled using the Lorenz-Mie theory enables investigations into the minute variations in the refractive index and size resulting from changes in local temperature and pollen aging.

Intraspecific variation in morphology of spiny pollen grains along an altitudinal gradient in an insect-pollinated shrub.

Intraspecific variations in pollen morphological traits are poorly studied. Interspecific variations are often associated with pollination systems and pollinator types. Altitudinal environmental changes, which can influence local pollinator assemblages, provide opportunities to explore differentiation in pollen traits of a single species over short distances. The aim of this study is to examine intraspecific variations in pollen traits of an insect-pollinated shrub, Weigela hortensis (Caprifoliaceae), along an altitudinal gradient. Pollen spine phenotypes (length, number and density), pollen diameter, lipid mass (pollenkitt) around pollen grains, pollen production per flower and pollinator assemblages were compared at four sites at different altitudes. Spine length and the spine length/diameter ratio of pollen grains were greater at higher altitudes but not correlated with flower or plant size. Spine number and density increased as flower size increased, and pollen lipid mass decreased as plant size increased. Bees were the predominant pollinators at low-altitude sites whereas flies, specifically Oligoneura spp. (Acroceridae), increased in relative abundance with increasing altitude. The results of this study suggest that the increase in spine length with altitude was the result of selection favouring longer spines at higher-altitude sites and/or shorter spines at lower-altitude sites. The altitudinal variation in selection pressure on spine length could reflect changes in local pollinator assemblages with altitude.

Quantifying the Influence of Pollen Aging on the Adhesive Properties of Hypochaeris radicata Pollen.

Although pollination is one of the most crucial biological processes that ensures plant reproduction, its mechanisms are poorly understood. Especially in insect-mediated pollination, a pollen undergoes several attachment and detachment cycles when being transferred from anther to insect and from insect to stigma. The influence of the properties of pollen, insect and floral surfaces on the adhesion forces that mediate pollen transfer have been poorly studied. Here, we investigate the adhesive properties of Hypochaeris radicata pollen and their dependence on pollen aging by quantifying the pull-off forces from glass slides using centrifugation and atomic force microscopy. We found that the properties of the pollenkitt-the viscous, lipid liquid on the surface of most pollen grains-influences the forces necessary to detach a pollen from hydrophilic surfaces. Our results show that aged H. radicata pollen form weaker adhesions to hydrophilic glass than fresh ones. On the other hand, when a pollen grain ages in contact with glass, the adhesion between the two surfaces increases over time. This study shows for the first time the pollen aging effect on the pollination mechanism.

Fresh "Pollen Adhesive" Weakens Humidity-Dependent Pollen Adhesion.

This study presents a quantitative investigation of pollen adhesion mediated by pollenkitt using pollen grains in their native state. Here, we attempt to clarify whether the exposure time, pollenkitt losses, and the surrounding humidity (relative humidity, RH) levels influence pollen adhesion. Pollen grains of Hypochaeris radicata (Asteraceae) were tested using atomic force microscopy. Regardless of the pollen condition (fresh, aged, and without pollenkitt), higher RH significantly increased pollen adhesion on hydrophilic surfaces, whereas it had little effect on pollen adhesion on hydrophobic surfaces. On hydrophilic surfaces, adhesion of fresh pollen was less dependent on RH than that of aged pollen or without pollenkitt, resulting in reduced adhesion under high RH. On hydrophobic surfaces, adhesion of fresh pollen was significantly lower than that of aged pollen. We utilized capillary models to explain the counterintuitive results obtained and came to the conclusion that the abundant fresh pollenkitt, which is widely accepted as pollen adhesive, can reduce pollen adhesion in some conditions. This study sheds light on the little-known adhesive properties of pollen and on the pollination mechanics.

Pollenkitt of some monocotyledons: lipid composition and implications for pollen germination.

The composition of pollenkitt and its role in the progamic phase of reproduction are poorly understood. With the aim of extending knowledge on these topics, we chose to study two monocotyledons rich in pollenkitt, with bi-celled and long-lived pollen and dry-type stigma: Crocus vernus Hill subsp. vernus and Narcissus poeticus L. Fatty acids of pollenkitt were assayed with gas chromatography. Germination tests were performed in vivo by pollinating the stigmas with a beard hair under a stereomicroscope, and in vitro in liquid culture medium using pollen, either treated or not, with carbon disulphide to remove pollenkitt. The pollen tube percentages were evaluated using fluorescence microscopy techniques. Scanning electron microscopy was used to examine pollen and to follow the early post-pollination stages. Pollenkitt forms bridges between pollen grains but not between grains and stigma papillae. It consists of a mixture of 25 fatty acids, most with long and unsaturated chains, among which are some omega acids. The same acids with different percentages persist on the peritapetal membrane. After its removal, the pollen loses adhesiveness and dries quickly, but retains full capacity for germination on the papillae and can even trigger germination in contiguous pollen grains that do not touch the papillae. The results, while confirming the key role of pollenkitt in protecting pollen and favouring pollination, suggest secondary roles in the progamic phase, and highlight the interactive ability of the pollen regardless of lipid shell. The predominance of fatty acids with 18:3 and 16:0, as already noted in Brassica napus pollenkitt, suggests their hierarchy independent of plant species.

Chemical modification of coating of Pinus halepensis pollen by ozone exposure.

Pollen coating, located on the exine, includes an extractible lipid fraction. The modification of the pollen coating by air pollutants may have implications on the interactions of pollen with plant stigmas and human cells. Pinus halepensis pollen was exposed to ozone in vitro and the pollen coating was extracted with organic solvent and analyzed by GC-MS. Ozone has induced chemical changes in the coating as observed with an increase in dicarboxylic acids, short-chain fatty acids and aldehydes. 4-Hydroxybenzaldehyde was identified as the main reaction product and its formation was shown to occur both on native pollen and on defatted pollen. 4-Hydroxybenzaldehyde is very likely formed via the ozonolysis of acid coumaric-like monomers constitutive of the sporopollenin. Modification of pollen coating by air pollutants should be accounted for in further studies on effect of pollution on germination and on allergenicity.

Micromorfología y ultraestructura de las anteras y los granos de polen en diez genotipos élite de Theobroma cacao (Malvaceae) / Micromorphology and ultrastructure of anthers and pollen grains in ten elite genotypes of Theobroma cacao (Malvaceae)

Resumen Introducción: A pesar de que T. cacao es una especie importante a nivel mundial por la producción de cacao, es poco lo que se conoce sobre la micromorfología y estructura de las anteras y los granos de polen. Objetivos: Describir y analizar la estructura y micromorfología de las anteras y los granos de polen de 10 genotipos élite de esta importante especie tropical. Métodos: Se tomaron más de 30 anteras de flores en antesis de los 10 genotipos élite de T. cacao del banco de germoplasma ex situ del Centro de Investigaciones Suiza-Agrosavia (Rionegro, Santander-Colombia). El material se procesó de acuerdo con los protocolos estándar para embeber y seccionar en parafina. Las secciones obtenidas (3 μm) se tiñeron con azul de Safranina-Alcian para discriminar estructuras con paredes primarias y secundarias y polifenoles totales. Además, se usó la técnica PAS-Amidoblack para diferenciar entre polisacáridos estructurales y de reserva, así como proteínas. Para la determinación de esporopolenina y polifenoles se usó la tinción azul de toluidina y finalmente para descripciones adicionales se aplicó la tinción azul alcián-PAS-hematoxilina. Las observaciones se realizaron mediante microscopio fotónico y microscopio de epifluorescencia. Para la observación con microscopía electrónica de barrido (MEB), las anteras con los granos de polen se fijaron y deshidrataron en 2.2 dimetoxipropano, luego se desecaron hasta un punto crítico y finalmente se recubrieron con oro. Resultados: Las anteras son bitecas y están sostenidas por un largo filamento formado por un estrato epidérmico, tejido parenquimatoso y un haz vascular. La dehiscencia ocurre longitudinalmente a través del estomio. La pared de la antera madura está formada por una capa epidérmica monoestratificada, una capa de células endoteliales con engrosamientos fibrilares lignificados y se pueden apreciar restos celulares del tapete y abundantes orbículas recubriendo la cavidad de los microesporangios. Los tejidos epidérmicos y parenquimatosos de las anteras almacenan polifenoles. Las orbículas son generalmente esféricas, psiladas y exhiben las mismas reacciones de tinción y fluorescencia que la exina de los granos de polen. Los granos de polen son mónades, isopolares, pequeños (16-19 µm) con amb circular, esferoidales, tricolpados con colpos medianos o cortos (5-10 µm) con membrana ornamentada, semitectatos, reticulados, heterobrochados, las paredes del retículo ornamentadas o no, con microgránulos de diferente tamaño o escabrados. Los análisis estadísticos mostraron que existen diferencias significativas en el tamaño de los granos de polen (P ˂ 0.05). Se observa que los granos de polen más pequeños son los del genotipo TCS 19 (16.890 µm) y se diferencian del resto de genotipos, y entre estos no se observan diferencias significativas. Solo dos genotipos (SCC 19 y SCA 6) presentaron polenkit y solo uno tiene paredes perforadas (SCA 6). Conclusiones: La estructura y micromorfología de las anteras de T. cacao son similares a las descritas para otras Malvaceae. Así mismo, los granos de polen mostraron variaciones de tamaño, ornamentación de las paredes y del lumen del retículo y presencia de polenkit. Sin embargo, no se observó relación entre las variaciones de los caracteres micromorfológicos analizados en los granos de polen y los modelos de compatibilidad polínica reportados para estos genotipos.

Abstract Introduction: Despite the fact that T. cacao is an important species worldwide for cocoa production, little is known about the micromorphology and structure of anthers and pollen grains. Objectives: To describe and analyze the structure and micromorphology of the anthers and pollen grains of 10 elite genotypes of this important tropical species. Methods: More than 30 anthers of flowers in anthesis were taken of the 10 elite genotypes of T. cacao from the ex situ germplasm bank of the Suiza-Agrosavia Research Center (Rionegro, Santander-Colombia). The anthers with the pollen grains were fixated and processed according to the standard protocols for embedding and sectioning in paraffin. Sections obtained (3 μm thick) were stained with Safranin-Alcian blue to discriminate structures with primary and secondary walls and total polyphenols. Additionally, the samples were also stained with the PAS-Amidoblack technique was used to differentiate between structural and reserve polysaccharides as well as proteins. Toluidine blue staining was used for the determination of sporopollenin and polyphenols and finally Alcian blue-PAS-Hematoxylin staining was applied for additional descriptions. Observations were made using photonic microscopy and epifluorescence microscopy. For observation with scanning electron microscopy (SEM) the anthers with the pollen grains were fixed and dehydrated in 2.2 Dimethoxypropane, then desiccated to critical point and finally coated with gold. Results: The anthers are dithecal and supported by a long filament made up of an epidermal stratum, parenchymal tissue, and a vascular bundle. The dehiscence occurs longitudinally through the stomium. The anther wall is made up of a monostratified epidermal layer, followed by a layer of endothecial cells with lignified fibrillar thickenings, cellular remnants of tapetum and abundant orbicules can be seen covering the cavity of the microsporangia. The epidermal and parenchymal tissues of the anthers are abundant in polyphenols. Orbicules are generally spherical, psilated, and these exhibit the same staining and fluorescence reactions as exine from pollen grains. The pollen grains are monades, isopolar, small (16-19 µm) with circular amb, spheroidal, tricolpate with medium or short colpi (5-10 µm) with sculptured membrane, semitectate, reticulated, heterobrochate, sculptured or non- sculptured walls, with microgranules of different size or scabrate. The statistical analyzes showed that there are significant differences in the size of the pollen grains (P ˂ 0.05). It is observed that the smallest pollen grains are those of the TCS 19 genotype (16.890 µm) and are different from the other genotypes, and among these there are no significant differences. Only two genotypes (SCC 19 and SCA 6) showed pollenkit and only one has perforated walls (SCA 6). Conclusions: The structure and micromorphology of the anthers of T. cacao are similar to those described for other Malvaceae. Likewise, the pollen grains showed variations in size, ornamentation of the sporoderm and the lumen of the reticulum and the presence of pollenkitt. However, no relationship was observed between the micromorphological characters analyzed in the pollen grains and the pollen compatibility models reported for these genotypes.

Effect of water absorption on pollen adhesion.

Pollens possess a thin liquid coating, pollenkitt, which plays a major role in adhesion by forming capillary menisci at interfaces. Unfortunately, the influence of humidity on pollenkitt properties and capillary adhesion is unknown. Because humidity varies widely in the environment, the answers have important implications for better understanding plant reproduction, allergy and asthma, and pollen as atmospheric condensation nuclei. Here, pollenkitt-mediated adhesion of sunflower pollen to hydrophilic and hydrophobic surfaces was measured as a function of humidity. The results quantify for the first time the significant water absorption of pollenkitt and the resulting complex dependence of adhesion on humidity. On hydrophilic Si, adhesion increased with increasing RH for pollens with or without pollenkitt, up to 200nN at 70% RH. In contrast, on hydrophobic PS, adhesion of pollenkitt-free pollen is independent of RH. Surprisingly, when pollenkitt was present adhesion forces on hydrophobic PS first increased with RH up to a maximum value at 35% RH (∼160nN), and then decreased with further increases in RH. Independent measurement of pollenkitt properties is used with models of capillary adhesion to show that humidity-dependent changes in pollenkitt wetting and viscosity are responsible for this complex adhesion behavior.

THE PROCESSES OF ANTHER DEHISCENCE AND POLLEN DISPERSAL: II. THE FORMATION AND THE TRANSFER MECHANISM OF POLLENKITT, CELL-WALL DEVELOPMENT OF THE LOCULUS TISSUES AND A FUNCTION OF ORBICULES IN POLLEN DISPERSAL.

The development of the locule of Gasteria verrucosa (Mill.) H. Duval and Lilium hybrida cv. Enchantment, especially the border between the sporophyte and the gametophyte, is investigated by means of light and electron microscopy, histochemistry and micromanipulation and related to pollen dispersal. The pecto-cellulosic cell walls of both the middle layer and the tapetum and a part of the endothecium disappear, and the tapetum cells are covered with sporopollenin-containing tapetal membranes and orbicules. The cell contents of the tapetum turn into the hydrophobic pollenkitt. In the locule, similar changes are observed as the pecto-cellulosic and callose walls of the meiocytes disappear and the future pollen grains are covered with the sporopollenin-containing exine. Due to their non-wettability, these hydrophobic substances on both sides of the sporophyte-gametophyte surface are important in pollen dispersal. The transfer of pollenkitt from the tapetum to the locule appears to be due to capillary forces in the locule after the continuous expansion of the pollen, rather than to active flow or to direct attraction by the pollen grains.

Role of flower and pollen aromas in host-plant recognition by solitary bees.

The means by which newly emerged pollen-specific (oligolectic) solitary bees locate their appropriate pollen host plant is not clearly understood. To evaluate the role of flower and pollen odors in this recognition process, preference tests were performed on foraging-inexperienced and, for comparison, field-experienced individuals of the solitary bee Colletes fulgidus longiplumosus. The bees were presented with a choice of four plant species, offered in the form of aromas from flowers, whole pollen, pollenkitt, and internal pollen lipids. Observed feeding-attempt response patterns suggest: 1) that bees can distinguish between plant species on the basis of odors from flowers, whole pollen, and pollenkitt, 2) inexperienced bees show a preference for the pollen on which they were reared as larvae, and 3) chemicals eliciting feeding responses to pollen appear to be contained in the pollenkitt. Feeding responses by experienced bees differ from those of inexperienced bees in ways which indicate that the insects' odor-based search image of the host plant becomes modified by foraging experience.