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.     

An improved cellophane method for in vitro germination of recalcitrant pollen

The cellophane technique of La Cour and Faberge has been improved by the use of a booklet of filter paper. The booklet consists of seven squares of filter paper stapled together; the cellophane on which the pollen is germinated is placed between the two top leaves of the booklet and the whole soaked in a sucrose-based nutrient medium for 15 min. This arrangement keeps the cellophane flat as it absorbs medium. The top leaf of the booklet is then removed, the pollen dusted on it and the completed preparation closed in a plastic-wrapped Petri dish. The lower leaves of the booklet keep the cellophane moist for up to 24 hr. Proportions of pollen grains germinating are at least as high as in the hanging drop method; pollen of species that germinate poorly or not all in hanging drops do well in this technique. Because the pollen tubes adhere tightly to the cellophane, staining, observation, and studies of various sorts are facilitated.     

An Improved Cellophane Method for in Vitro Germination of Recalcitrant Pollen

The cellophane technique of La Cour and Faberge has been improved by the use of a booklet of filter paper. The booklet consists of seven squares of filter paper stapled together; the cellophane on which the pollen is germinated is placed between the two top leaves of the booklet and the whole soaked in a sucrose-based nutrient medium for 15 min. This arrangement keeps the cellophane flat as it absorbs medium. The top leaf of the booklet is then removed, the pollen dusted on it and the completed preparation closed in a plastic-wrapped Petri dish. The lower leaves of the booklet keep the cellophane moist for up to 24 hr. Proportions of pollen grains germinating are at least as high as in the hanging drop method; pollen of species that germinate poorly or not all in hanging drops do well in this technique. Because the pollen tubes adhere tightly to the cellophane, staining, observation, and studies of various sorts are facilitated.     

Calcium crystals in the anther of Petunia: the existence and biological significance in the pollination process

Using an X-ray microanalysis system fitted with variable-pressure scanning electron microscopy, we noted that many calcium crystals accumulated under the stomium in the anther of Petunia. When the anther was dehisced and pollen grains were released from the stomata, the calcium crystals adhered to pollen grains and moved to the stigma together with pollen grains. In contrast, an X-ray microanalysis of the stigma surface before pollination detected no calcium emission on the stigma surface. Furthermore, pollen germination and pollen tube growth in medium without Ca occurred as in complete medium. However, after the pollen grains had been washed with abundant germination medium without calcium, pollen germination in the medium without Ca was inhibited. These results show that the calcium crystals dissolved in the aqueous drop under the exudate on the stigma and supplied calcium ions for pollen germination. In addition, calcium crystals were produced not only in the anther of Petunia but also in Nicotiana, suggesting that calcium crystals supply pollen grains with the calcium ions required for pollen germination and serve to improve reproduction efficiency in Solanaceae.     

Thin-Layer Lactose Agar for Pollen-Tube Culture of Tradescantia To Enhance Planar Distribution of Chromosomes

Mature pollen grains of Tradescantia paludosa were sown on a thin layer of lactose-agar medium at 38-39 C. After 16 hr incubation, slides were fixed in aceto-alcohol (1:3; for 1-3 hr, hydrolyzed in 1 N HC1 at 60 C, and treated with water at 65 C. Delamination of the upper layer of medium was accomplished in cold water. The delaminated upper layer of solidified medium containing most of the ungerminated pollen and underdeveloped pollen tubes was flushed off with running water. The remaining single layer of pollen tubes was flattened and firmly affixed to the slide by pressing under a coverglass on a hot surface at 80 C. The quick-freeze technique was used to remove the coverglass prior to staining, dehydration and permanent mounting. Preparations made according to this procedure gave a planar chromosome distribution and approximately 160 analyzable metaphase figures per slide, thus facilitating aberration analysis and autoradiography. Comparative studies on the effectiveness of colchicine, Colcemid, and acenapthene in arresting mitotic nuclei at metaphase indicated that Colcemid was more effective than the others, but that it caused deformities of the metaphase chromosomes and induced chromatid breaks at a concentration of 0.2 mg/ml or higher. Colchicine is a more favorable chemical than the other two.     

Directional Guidance of Nicotiana alata Pollen Tubes in Vitro and on the Stigma

Pollen tubes navigate the route from stigma to ovule with great accuracy, but the cues that guide them along this route are not known. We reproduced the environment on the stigma of Nicotiana alata by immersing pollen in stigma exudate or oil close to an interface with an aqueous medium. The growth of pollen in this culture system mimicked growth on stigmas: pollen grains hydrated and germinated, and pollen tubes grew toward the aqueous medium. The rate-limiting step in pollen germination was the movement of water through the surrounding exudate or oil. By elimination of other potential guidance cues, we conclude that the directional supply of water probably determined the axis of polarity of pollen tubes and resulted in growth toward the interface. We propose that a gradient of water in exudate is a guidance cue for pollen tubes on the stigma and that the composition of the exudate must be such that it is permeable enough for pollen hydration to occur but not so permeable that the supply of water becomes nondirectional. Pollen tube penetration of the stigma may be the most frequently occurring hydrotropic response of higher plants.     

Collodion Membranes in Pollen Tube Technique

Membranes are formed by allowing a drop of collodion-acetone solution to come into contact with the surface of warm sugar solution in a petri dish. Pollen is germinated upon the smooth areas of the membrane when all traces of acetone have evaporated. Semipermanent preparations are made by isolating the pollinated area of the membrane, floating it onto a slide, and, after the removal of excess sugar solution, adding a drop of acetic-stain fixative, followed by an albumenized cover slip. The preparation can be made permanent by inverting a slide in a mixture of 1 part glacial acetic acid and 3 parts absolute alcohol, when the collodion membrane will dissolve and allow the cover slip and adhering grains to fall free. The cover slip is then passed through absolute alcohol (2 changes), xylene, and mounted in neutral mountant on a clean slide. By substituting a drop of the alcohol-acetic acid mixture in place of acetic-stain fixative, the grains adhering to the cover slip may be stained by the Feulgen method.     

Studies on heteromorphic self-incompatibility systems: Physiological aspects of the incompatibility system of Primula obconica

Summary In Primula obconica, a species with a heteromorphic self-incompatibility system, the distinction between compatible and incompatible pollen tubes takes place on the stigma surface in thrum flowers, self tubes growing randomly over the papillar cells. No differences were seen between self and cross tube behaviour on the pin stigma surface, but self tubes were inhibited within the stigmatic tissue with differences in tube length evident after 24 h. The stigma surface bears a proteinaceous pellicle and binds the lectin Concanavalin A. Removal of the stigma removes the incompatibility barrier in mature gynoecia. Bud pollination shows that pollen tubes cannot grow in a normal manner on immature stigmas; the random growth of tubes over the stigma surface resembles that of mature thrum selfs. Fewer compatible tubes reach the style base of young gynoecia and smaller numbers of seeds are set than in mature flowers. Pin and thrum pollen grains germinate and grow in aqueous media, thrum tubes growing longer than pin. The presence of H₃BO₄ and CaCl₂ in the growth medium promotes tube elongation and lengths equivalent to compatible styles can be obtained. The pollen grains have proteinaceous materials in their walls which diffuse out on moistening. Prolonged washing in aqueous media removes these materials but the incompatibility reaction remains unchanged. Thus the incompatibility reaction is between pollen tubes and stigmatic tissue and differs from the homomorphic, sporophytic system where pollen wall proteins elicit the incompatibility response.     

The effect of varying the number of pollen grains used in fertilization

Summary The number of pollen grains placed upon a stigma influence both the development of pollen tubes and subsequently the progeny which result from fertilizations by gametes from these pollen tubes. The first influence is demonstrated by reduced pollen tube growth rates when pollen grains are few in number. This may indicate direct effects of pollen tubes upon the stylar tissues or perhaps more complex interactions between pollen and style. The second and potentially more important influence of limited pollination is upon the progeny. This was demonstrated with studies on three species. In each case, variation among the resultant plants was greater when pollen was limited than when normal, that is excessive, pollen was used. The mechanism of this phenomenon is not certain, but our data indicate that it is not simply an artefact of variation in seed size.     

紫萼花粉粒和花粉管中微丝的超微结构

用常规化学固定和化学固定前用鬼笔环肽处理两种电镜样品制作技术,分别研究了紫萼[Hosta venteicosa (=H.coerulea]成熟花粉粒和幼花粉管中的微丝的超微结构。结果表明,在常规电镜固定中花粉粒中的微丝能保存,但在花粉管中的则遭受破坏。用鬼笔环肽处理后化学固定的方法,微丝在花粉管中能良好地保存。在花粉粒中平行的微丝形成束,表现为具分布的特点,即限于分布在它们功能的区域,并且微丝束经常紧密地与营养核贴近。在幼花粉管中微丝束表现为在线粒体、质体、内质网、小泡和小液泡的表面通过,并常常与脂体紧密联结。这些现象表明在花粉萌发和花粉管生长时,微丝与营养核及与其它细胞器的运动之间存在某些联系的迹象。     

Ultrastructure of Microfilaments in Pollen and Pollen Tubes of Hosta Ventricosa (=H. voerulea)

Ultrastructure of microfilaments in pollen grains and pollen tubes of Hosta ventricosa (=H. coerulea) was investigated. Results indicate that microfilaments with conventional chemical fixation are preserved only in pollen grains, but destroyed in pollen tubes. Microfilaments treated with phalloidin before chemical fixation are found preserved in pollen tubes. In pollen grains a pronounced organization of parallel microfilaments appeared in bundles with its distribution characteristics is always restricted to their functional domains where bundles were in close contact with the vegetative nucleus. In young pollen tubes cytoplasmic bundles of microfilaments appeared also to pass close to the surface of mitochondria, plastids, endoplasmic reticulum, vesicles and small vacuoles, and always associated with lipid bodies. These findings strongly indicate that there is a relationship between microfilaments and the movement of vegetative nucleus and other organelles in the germination of pollen grains and in the growth of pollen tubes.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

Some permeability properties of angiosperm pollen grains,pollen tubes and generative cells

Summary The permeability of pollen grains, pollen tubes and generative cells of Helleborus foetidus and Galanthus nivalis has been investigated using four probes spanning a wide range of molecular weights: 4,6-diamidino-2-phenyl indole (DAPI; mol.wt. 350). Evans blue (mol.wt. 960), FITC-dextran (average mol.wt. 19400) and FITC-albumin (average mol.wt. 67000). DAPI penetrated into the vegetative cells of desiccated and hydrated pollen, and also entered growing pollen tubes. In contrast, the generative cells of hydrated pollen and of pollen tubes were highly resistant to penetration, as they were when isolated in osmotically balancing medium. Evans blue failed to enter intact generative cells under any of the conditions tested. The dye ultimately entered the vegetative cells of some pollen grains, but these were non-germinable. Growing pollen tubes invariably resisted penetration. Neither of the high molecular weight conjugates entered germinable pollen grains or intact pollen tubes. The results suggest that it is highly unlikely that DNA fragments of high molecular weight can enter viable pollen, pollen tubes or generative cells under any normal conditions.     

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]。     

The Germination of Vinca rosea L. Pollen Grains and the Growth of the Pollen Tubes in vitro

The effect of medium concentration, pollen grain concentration, pH of the media, light and temperature on the germination of Vin ca rosea pollen grains, and the growth of their pollen tubes in vitro have been studied. The pollen grains germinate best at a sucrose concentration between 14.2% and 30%; when the pollen grain concentration exceeds 800 per 0.0234 ml; at near neutral pH (6.5); in darkness and at a temperature close to 30°. Moreover buffering ions affect the growth of the pollen tubes. Pollen grains remain viable in a wide range of temperatures, and the wall of the pollen grain is capable of withstanding severe osmotic imbalance. Low temperature induces spherical swellings at the tips of the pollen tubes, followed by accumulation of a hyaline plug.     

Differential roles of microtubule and actin-myosin cytoskeleton in the growth of Pinus pollen tubes

The behavior and role of the microtubule (MT) and actin-myosin components of the cytoskeleton during pollen tube growth in two species of Pinus were studied using anti--tubulin, rhodamine-phalloidin, anti-myosin, and the appropriate inhibitors. Within germinated pollen tubes MTs were arranged obliquely or transversely, but in elongated tubes they were arranged along the tube's long axis. MTs were localized in the tube tip region, excluding the basal part. Altered growth was found in pollen tubes treated with colchicine; the tips of many pollen tubes incubated in the liquid medium were branched and/or rounded, and those in the agar medium were divided into many branches. Both the branching and the rounding were considered to be caused by the disturbance of polarizing growth of the tube due to MT disorganization with colchicine treatment. Actin filaments (F-actin) were found in the major parts of many pollen tubes along their long axis, excluding the tip region. In a few tubes, however, F-actin was distributed throughout the tube. The areas in the pollen tube containing F-actin were filled with abundant cytoplasmic granules, but the areas without F-actin had very few granules. The tube nucleus, which migrated from the grain area into the tube, was closely associated with F-actin. Germination of pollen grains treated with cytochalasin B was little affected, but further tube elongation was inhibited. Myosin was identified on cytoplasmic granules and to a lesser extent on the tube nucleus in the pollen tubes. Several granules were attached to the nuclear envelope. Tube growth was completely inhibited by N-ethylmaleimide treatment. In generative cells that were retained in the pollen grain, both MT and F-actin networks were observed. Myosin was localized on the cytoplasmic granules but not on the cell surface. In conclusion, it was shown that actin-myosin and MTs were present in gymnospermous Pinus pollen tubes and it is suggested that the former contributed to outgrowth of the tubes and the latter contributed to polarized growth. Several differences in the behavior of cytoskeletal elements in generative cells compared to angiosperms were revealed and are discussed.     

The distribution of membrane-bound 14-3-3 proteins in organelle-enriched fractions of germinating lily pollen

Proteins of the 14-3-3 family show a broad range of activities in plants, depending on their localisation in different cellular compartments. Different organelle membranes of pollen grains and pollen tubes of Lilium longiflorum Thunb. were separated simultaneously using optimised discontinuous sucrose density centrifugation. The obtained organelle-enriched fractions were identified as vacuolar, Golgi, endoplasmic reticulum and plasma membranes, according to their marker enzyme activities, and were assayed for membrane-bound 14-3-3 proteins by immunodetection. 14-3-3 proteins were detected in the cytoplasm as well as in all obtained organelle fractions but were also released into the extracellular medium. In pollen grains, much more plasma membrane-bound 14-3-3 proteins were detected than in the PM-enriched fraction of pollen tubes, whereas the level of Golgi- and ER-associated 14-3-3 proteins was similar in pollen grains and tubes. This shift in the localisation of membrane-associated 14-3-3 proteins is probably correlated with a change in the major function of 14-3-3 proteins, e.g., perhaps changing from initiating pollen grain germination by activation of the PM H +-ATPase to recruitment of membrane proteins via the secretory pathway during tube elongation.     

Pollen morphology and germination inNarcissus

Pollen grains of several species and varieties ofNarcissus were examined with a light microscope and a scanning electron microscope. Normal pollen grains were kidney- or spindle-shaped with a germination furrow and a reticulate structure similar to that of the pollen grains ofAmaryllis. Pollen grains germinated within 2 to 3 hr. Percentage of germination was dependent upon temperature and treatment. Pollen tubes grew in length up to 1,000 μm and branched occasionally or behaved in strange fashion. Fresh pollen grains germinated more in distilled water at lower temperature than in sucrose-aqueous medium. Both in the presence and in the absence of stigmatic exudate calcium increased the percentage of germination. Gibberellic acid, abscisic acid and coumarin inhibited the pollen germination. Plasmoptysis occurred in all species and in all media tested except in a medium containing coumarin without stigmatic exudates. Plasmoptysis did not seem to be induced by hypotonic medium alone. Pollen of high germination capacity showed a high percentage of plasmoptysis. Based on the results obtained, evolution and sterility of theNarcissus plant was discussed.     

Division in Isolated Generative Cells of Allemanda neriilolia

An osmotic shock method of isolating generative cells from Allemanda neriifolia was described. Fresh pollen grains were first placed ill a Brewbaker and Kwack's medium (BK medium) containing 50% sucrose, incubated at 28℃ for 2 hours. During this incubation period pollen grains germinated and produced pollen tubes measuring about 200 μm long. After this initial incubation period, a fixed amount of BK medium without sucrose was added thus diluting the original medium to a sucrose concentration of 30% – an optimum concentration for generative cell growth. The addition, of the BK medium without sucrose brought about an osmotic shock effect on the pollen tubes and caused most of the tubes to burst at the tip region thus releasing the contents together with the generative cell from the tube into the 30% sucrose + BK medium. After isolation and filtering into a fresh lot of 30% sucrose + BK medium, generative cells changed from spindle into spherical-shaped cells. In the 30% sucrose + BK medium, the generative cells divided and within a short period of 3 to 5 hours a laege number of cells at various stages of mitosis was obtained.     

Gametogenesis of tobaccoin vitro

Excised anthers ofNicotiana tabacum L. were culturedin vitro at the stage of pollen tetrads, which proved in our experiments to be the most suitable initial stage for cultivation, up to the stage of mature pollen grains, using Ito and Stern's nutrient medium, or this medium supplemented with uracil. Germinating capacity of the pollen grains formed and the lengths of pollen tubes were quantitatively evaluated.