Lactic Acid Clearing and Fluorescent Staining for Demonstration of Sieve Tubes

Sieve tubes of the phloem in cleared plant parts can be located by means of a staining reaction specific for callose. The plant part is decolourized in 1:3 glacial acetic acid-95% ethanol and cleared in hot 85% lactic acid at 98-100 C. Callose is not dissolved by this treatment and is then stained with 0.01% analine blue in 0.07 M phosphate buffer, pH 7.5, and observed by fluorescence microscopy. A sieve tube is recognized by the bright yellow fluorescence of the callose on its sieve plates. In most tissues, a natural light yellow fluorescence of the parenchyma cells is evident after the clearing step. This intensifies upon staining with analine blue and tends to make the tissue opaque, but it can be minimized by quick-killing of the tissue before commencing the decolourization. The procedure gives best results when applied to young tissues in which interference from the natural yellow fluorescence of lignified cells such as xylem elements and phloem fibers is minimal. Callose plugs in pollen tubes were also shown in intact, cleared styles.     

The division of the generative nucleus and the formation of callose plugs in pollen tubes of Aechmea fasciata (Bromeliaceae) cultured in vitro

The effect of different external factors on pollen germination and pollen tube growth is well documented for several species. On the other hand the consequences of these factors on the division of the generative nucleus and the formation of callose plugs are less known. In this study we report the effect of medium pH, 2-[N-morpholino]ethanesulfonic acid (MES) buffer, sucrose concentration, partial substitution of sucrose by polyethyleneglycol (PEG) 6000, arginine (Arg), and pollen density on the following parameters: pollen germination, pollen tube length, division of the generative nucleus, and the formation of callose plugs. We also studied the different developmental processes in relation to time. The optimal pH for all parameters tested was 6.7. In particular, the division of the generative nucleus and callose plug deposition were inhibited at lower pH values. MES buffer had a toxic effect; both pollen germination and pollen tube length were lowered. MES buffer also influenced migration of the male germ unit (MGU), the second mitotic division, and the formation of callose plugs. A sucrose concentration of 10% was optimal for pollen germination, pollen tube growth rate and final pollen tube length, as well as for division of the generative nucleus and the production of callose plugs. Partial substitution of sucrose by PEG 6000 had no influence on pollen germination and pollen tube length. However, in these pollen tubes the MGU often did not migrate and no callose plugs were observed. Pollen tube growth was independent of the migration of the MGU and the deposition of callose plugs. In previous experiments Arg proved to be positive for the division of the generative nucleus in pollen tubes cultured in vitro. Here, we found that more pollen tubes had callose plugs and more callose plugs per pollen tube were produced on medium with Arg. After the MGU migrated into the pollen tube (1 h after cultivation), callose plugs were deposited (3 h). After 8 h the first sperm cells were produced. The MGU moved away from the active pollen tube tip until the second pollen mitosis occurred, thereafter the distance from the MGU to the pollen tube tip diminished. Callose plug deposition never started prior to MGU migration into the pollen tube. Pollen tubes without a MGU also lack callose plugs (±30% of the total number of pollen tubes). Furthermore, we found a correlation between the occurrence of sperm cells in pollen tubes and the synthesis of callose plugs.     

Observations of pollen tube growth in Nicotiana alata and their implications for the mechanism of self-incompatibility

 Style squashes and stylar grafts were used to examine the growth of Nicotiana alata pollen tubes in self-compatible and self-incompatible styles. Compatible tubes typically showed a uniform layer of callose deposition in the walls and in small plugs spaced at regular intervals within the tube. Incompatible tubes were characterised by the variability of callose deposition in the walls and by larger, closer and more irregularly spaced plugs. There was no difference in the growth rate of compatible and incompatible tubes during growth through the stigma, but within the style most compatible tubes grew 20–25 mm day^-1 (maximum 30 mm day^–1), whereas incompatible tubes grew 1.0–1.5 mm day^-1 (maximum 5 mm day^–1). Many incompatible tubes continued to grow until flowers senesced, and only a small proportion died as a consequence of tip bursting. Grafting compatibly pollinated styles onto incompatible styles showed that the incompatible reaction could occur in pollen tubes between 2 and 50 mm long, and that inhibition of pollen tube growth occurred in both the upper and lower parts of the transmitting tract. Grafting incompatibly pollinated styles onto compatible styles showed that the incompatible reaction was fully reversible in at least a proportion of the pollen tubes. The findings are not consistent with the cytotoxic model of inhibition of self-pollen tubes in solanaceous plants, which assumes that the incompatible response results from the degradation of a finite amount of rRNA present in the pollen tube. However, if pollen tubes do in fact synthesise rRNA, the findings become consistent with this model. Received: 23 May 1996 / Revision accepted: 22 August 1996     

Staining and Observing Pollen Tubes in the Style by Means of Fluorescence

Pollen tubes in the styles of the tomato and of other flowering plants can be observed by using the following technic. Styles are fixed in formalin-acetic-80% alcohol (1:1:8) and cleared and softened in a strong (8 N) sodium hydroxide solution. Staining is accomplished in a 0.1% solution of water-soluble aniline blue dye dissolved in 0.1 N, K₃PO₄. The styles are smeared or are observed whole under a conventional or dissecting microscope by direct illumination with ultraviolet light of a wavelength of about 356 m°. Observations are made in a darkened room. Under these conditions callose fluoresces bright yellow-green and contrasts strongly with the bluish or grayish fluorescence of the stylar tissue. The pollen tubes are outlined by a callose lining and irregularly spaced callose plugs.     

Applications of fluorochromes to pollen biology. II. The DNA probes ethidium bromide and Hoechst 33258 in conjunction with the callose-specific aniline blue fluorochrome

Techniques are described for detection of pollen grain and pollen tube nuclei using the fluorescent DNA probes ethidium bromide or Hoechst 33258, in conjunction with the aniline blue fluorochrome sirofluor, which stains the callose component of pollen tube walls and plugs. The DNA probes, which may be used either as vital stains or following fixation, permit discrimination between vegetative and generative or sperm nuclei. Double staining with sirofluor allows location of nuclei within pollen tubes grown in vitro, and when used after pollination enables the viewer to discriminate between nuclei within the pollen tube vs. nuclei of the pistil tissue.     

Applications of Fluorochjromes to Pollen Biology. Ii. The Dna Probes Ethidium Bromide and Hoechst 33258 in Conjunction with the Callose-Specific Aniline Blue Fluorochrome

Techniques are described for detection of pollen grain and pollen tube nuclei using the fluorescent DNA probes ethidium bromide or Hoechst 33258, in conjunction with the aniline blue fluorochrome sirofluor, which stains the callose component of pollen tube walls and plugs. The DNA probes, which may be used either as vital stains or following fixation, permit discrimination between vegetative and generative or sperm nuclei. Double staining with sirofluor allows location of nuclei within pollen tubes grown in vitro, and when used after pollination enables the viewer to discriminate between nuclei within the pollen tube vs. nuclei of the pistil tissue.     

Studies on growth and utilization of stylar carbohydrate by pollen tubes and callose development in self-incompatibleOenothera organesis

The addition of boric acid or calcium, or both boric acid and calcium to the warm water in which the styles were treated led to a further enhancement of pollen tube growth in otherwise incompatible styles. It was established that developing pollen tubes ofOenothera organesis obtain sugar from the stylar tissue through which they grow. The rate of this utilization of endogenous sugar was stimulated by the addition of boric acid. It was further established that with increased pollen tube development there was reduction in callose plugs as well as in content of endogenous stylar sugar.     

Immunocytochemical localization of callose in the germinated pollen ofCamellia japonica

Summary A polyclonal antibody against -1,3-glucan, callose, extracted from the pollen tube wall ofCamellia japonica was raised in mice and, using it as a probe, the localization of callose in the germinated pollen was studied. By confocal laser scanning microscopy, callose was found in the tip region of the pollen tube and the tube wall; the immuno-fluorescence in the tube wall was less toward the base of the tube. In contrast, the tip region did not fluoresce although the whole of the tube wall did strongly with aniline blue, the specific dye for callose. Immuno-electron microscopy showed that callose was also found in Golgi vesicles which concentrated in the tip region of the pollen tube, the inner layer of the tube wall, callose plugs, and Golgi vesicles in the pollen grain. Immuno-gold labeling was often detected on the fibrous structures in Golgi vesicles and callose plugs. Based on these results, the participation of Golgi vesicles in the formation of the tube wall and callose plugs was discussed.Abbreviation TBS Tris-buffered saline - Tris Tris(hydroxy-methyl)-aminomethane - PBS phosphate-buffered saline - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - CLSM confocal laser scanning microscopy - DP degree of polymerization     

从水稻花粉管分离精细胞(简报)

精细胞的分离是植物生殖工程的一个重要组成部分,是目前被子植物有性生殖研究的一个活跃领域[1,2]。随着精细胞分离技术的完善和分离出精细胞的植物类型的增加,目前对精细胞的分子生物学研究已有一些进展,主要是精细胞特异蛋白的分离[3,4]和cDNA文库的构建以及一些精细胞特异基因的分离[5,6]。     

Effect of pollen-style interaction on the pollen tube growth of Gossypium hirsutum

Summary All possible crosses among 5 strains of Gossypium hirsutum were made, and the pollen tubes were grown in vivo for 4 h before being fixed, stained and measured. Temperatures ranging from 18.5 to 40.0 °C were tested for pollen germination and pollen tube growth. The optimal temperature for pollen tube growth was 30.0 °C. Relative humidity levels of 0 to 100% were used as a pre-pollination treatment of the pollen. Significant differences among the mean pollen tube length of the strains occurred due to pollenXstyle interactions. The strains also differed in the number of styles which did not support pollen tube growth. These differences were also due to pollenXstyle interactions. Pollen and style strains could be ranked according to their relative contribution to pollen tube length.College of Agricultural Sciences Publication Number T-4-189     

Pollen performance before and during the autotrophic-heterotrophic transition of pollen tube growth

For species with bicellular pollen, the attrition of pollen tubes is often greatest where the style narrows at the transition between stigmatic tissue and the transmitting tissue of the style. In this region, the tubes switch from predominantly autotrophic to predominantly heterotrophic growth, the generative cell divides, the first callose plugs are produced, and, in species with RNase-type self-incompatibility (SI), incompatible tubes are arrested. We review the literature and present new findings concerning the genetic, environmental and stylar influences on the performance of pollen before and during the autotrophic-heterotrophic transition of pollen tube growth. We found that the ability of the paternal sporophyte to provision its pollen during development significantly influences pollen performance during the autotrophic growth phase. Consequently, under conditions of pollen competition, pollen selection during the autotrophic phase is acting on the phenotype of the paternal sporophyte. In a field experiment, using Cucurbita pepo, we found broad-sense heritable variation for herbivore-pathogen resistance, and that the most resistant families produced larger and better performing pollen when the paternal sporophytes were not protected by insecticides, indicating that selection during the autotrophic phase can act on traits that are not expressed by the microgametophyte. In a study of a weedy SI species, Solanum carolinense, we found that the ability of the styles to arrest self-pollen tubes at the autotrophic-heterotrophic transition changes with floral age and the presence of developing fruits. These findings have important implications for selection at the level of the microgametophyte and the evolution of mating systems of plants.     

Recent Advances in Microtechnic. I. Methods of Studying the Development of the Male Gametophyte in Angiosperms

Among methods used for a study of nuclear details in the development of pollen grains, the following were found to be very satisfactory: (1) warming the entire grains in aceto-carmine and then clearing with chloral hydrate; (2) making smear preparations stained with crystal-violet-iodine or iron alum hematoxylin. For paraffin sections, a counterstain with dilute alcoholic erythrosin is often very useful after the usual iron hematoxylin technic.

A method of making cultures of pollen tubes on slides coated with thin films of sugar agar is described in detail. The tubes can be fixed by immersing the slide in formol-acetic-alcohol and then stained by any desired schedule. Iron alum hematoxylin was found to be the most satisfactory, but the Feulgen reaction is very valuable in such cases where the nuclei are obscured by the density of the pollen tube cytoplasm. Living pollen tubes can be kept under observation by dissolving a small quantity of neutral red or other vital stain in the sugar agar before it is spread on the slide.

For studying stages in fertilization or gametogenesis, styles should be fixed and sectioned only after a preliminary study with iodine-chloral-hydrate or safranin-anilin-blue or aceto-carmine. Once the extent to which pollen tubes grow in a given time in the stylar tissues has been determined, it is possible to fix material with some knowledge of what it is going to show.

Some other methods, that have not been tried by the authors but appear to be valuable, are also briefly described.     

十字花科10属种与油菜萝卜胞质不育系杂交的花粉萌发情况观察

对10属种十字花科植物与油菜萝卜胞质不育系杂交时花粉在柱头上粘合、萌发、花粉管伸长等情况进行观察。结果表明：（1）海甘蓝花粉粒粘合较难;（2）48 h内无瓣焊菜〖WTBX〗(Rorippa dubia)〖WTBX〗、毛果诸葛菜(Orychophragmus violaceus)、桂竹香(Cheiranthus cheiri)、海甘蓝(Crambe abyssinica)花粉管的伸长受阻于花粉萌发启动之时,花粉壁内形成胼胝质塞;播娘蒿、紫罗兰、荠菜花粉管伸长但未进入乳突细胞;芝麻菜花粉管进入乳突细胞而未进入柱头,‘浠水白’(Brassica campestris)、蓝花子有花粉管进入柱头及花柱而未进入胚囊。     

Movement of generative cell and vegetative nucleus in tobacco pollen tubes is dependent on microtubule cytoskeleton but independent of the synthesis of callose plugs

The role of microtubules (MTs) in vegetative nucleus (VN) and generative cell (GC) transport was investigated by comparing VN and GC distribution with callose plug formation in tobacco pollen grains germinated and grown for 12 h with the plant-specific anti-MT drug oryzalin. The VN-GC complex or VN alone was located close to the tube tip in 100% of controls, but in only 5% of oryzalin-treated tubes. Instead, in 38% of oryzalin tubes, the complex or VN occurred close to the last-formed callose plug; in 40% between or in the middle of plugs; and in 17%, in or near the grain. An aberrant microfilament (MF) cytoskeleton was revealed by expression of a green fluorescent protein-talin fusion protein in living oryzalin-treated tubes. The abnormal MF structures probably resulted from the absence of MTs and impaired - or were a consequence of - VN and GC movement into the tube tip. In oryzalin tubes with several callose plugs, the VN and GC could be in or near the grain, indicating that callose plug synthesis is not dependent on the movement of VN and GC into the tube. VN and GC movement and callose plug formation are apparently independent events, in which the transport of the VN-GC complex must precede callose plug synthesis. Maintenance of the correct developmental program requires an intact MT cytoskeleton, otherwise no fertile pollen tubes are formed.     

Evaluation of pollen tube growth ability in Petunia species having different style lengths

Pollen tube growth is essential for the fertilization process in angiosperms. When pollen grains arrive on the stigma, they germinate, and the pollen tubes elongate through the styles of the pistils to deliver sperm cells into the ovules to produce the seeds. The relationship between the growth rate and style length remains unclear. In previous studies, we developed a liquid pollen germination medium for observing pollen tube growth. In this study, using this medium, we examined the pollen tube growth ability in Petunia axillaris subsp. axillaris, P. axillaris subsp. parodii, P. integrifolia, and P. occidentalis, which have different style lengths. Petunia occidentalis had the longest pollen tubes after 6 h of culture but had a relatively shorter style. Conversely, the pollination experiments revealed that P. axillaris subsp. parodii, which had the longest style, produced the longest pollen tubes in vivo. The results revealed no clear relationship between the style lengths and the growth rate of pollen tubes in vitro. Interspecific pollinations indicated that the styles affected pollen tube growth. We concluded that, in vitro, the pollen tubes grow without being affected by the styles, whereas, in vivo, the styles significantly affected pollen tube growth. Furthermore, interspecific pollination experiments implied that the pollen tube growth tended to be suppressed in the styles of self-incompatibility species. Finally, we discussed the pollen tube growth ability in relation to style lengths.     

An in vitro assay for assessing the effects of growth factors on Nicotiana alata pollen tubes

 A new method for assessing the effects of test compounds on Nicotiana alata pollen tubes in culture is described. Pollen tubes grow from a cluster of grains placed beneath a thin layer of gelled medium in which test substances are incorporated and from which evaporation is prevented by a covering layer of oil. Pollen tubes can grow to 8 mm in length in 24 h, which corresponds to about 25% of the maximum growth rate in styles. Growth is non-destructively measured. The developmental stages reached by cultured tubes are similar to those of tubes growing in styles; growth changes from being reserve-dependent to reserve-independent, callose plugs form, and the nucleus of the generative cell divides. Because culture volumes are small (10–20 μl per replicate), the effects of known concentrations of microgram quantities of compounds on the growth of pollen tubes can be tested. Received: 25 February 1997 / 21 July 1997     

Cytochemical Analysis of Callose Localization in Lilium longiflorum Pollen Tubes

Callose, a ß, 1–3 glucan as a component of plantcells has received sporadic attention. Here, we report an attemptto determine whether aniline blue and lacmoid are indeed specificfor visualizing callose. We also re-evaluate, based on a checkfor stain specificity, the localization of callose in elongatingLilium longiflorum, cv. ‘Ace’ pollen tubes. Specificityof these stains was checked by chemical and enzymatic extractionprocedures which solubilize proteins and polysaccharides. Resultsherein question the generally accepted validity of the fluorescent-anilineblue method for detecting callose. Lacmoid either possessesan affinity for both callose and protein or for callose as aglycoprotein. As for callose localization, the walls of thenon-growing region of the lily pollen tube contain callose,probably as a glycoprotein. Presence of the callosicglycoproteinin the wall of the growing tube-tip is dependent on tube length.Callose plugs exhibiting an affinity for aniline blue or lacmoidwere never seen. Phase-contrast microscopy revealed non-stainablewall ingrowths in fixed-tubes and free-moving cytoplasmic masseswithin living tubes.     

Distribution of unesterified and esterified pectins in cell walls of pollen tubes of flowering plants

Immunocytochemical localization of polygalacturonic acid (pectin) and methyl-esterified pectin in the walls of pollen tubes of 20 species of flowering plants grown in vitro was investigated by using monoclonal antibodies (MAbs) JIM5 and JIM7 and by means of confocal laser scanning microscopy (CLSM). In general, periodic annular deposits of pectins were found coating the tube wall in species possessing solid styles, and a more uniform pectin sheath in tube walls in species having hollow styles or no styles. We hypothesize that the periodic ring-like structure of the pectin sheath reinforces pollen tubes for passing through the transmitting tract in the style. Esterified pectin which prevents Ca²⁺-induced gelification of pectate is located predominantly at the apex. This implies that pectin esterification is related to tip wall loosening that is required for cell wall expansion during tip growth of pollen tubes. The occurrence of unesterified pectins in other areas of pollen tube walls suggests that de-esterification of pectin following tip expansion leads to a more rigid form of pectin that contributes to the construction of the pollen tube wall.     

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