花粉管引导机制的研究进展

花粉管引导是指显花植物在受精过程中,雌蕊组织与花粉管相互作用使花粉管定向生长并最终到达胚囊的过程,其机制颇为复杂。该文基于调控花粉管生长的孢子体引导和配子体细胞引导两个主要过程,阐述雌蕊中不同蛋白分子和其它小分子物质的浓度梯度在花粉管的孢子体组织引导中的作用,以及胚囊中不同类型的细胞及其相关基因与蛋白在花粉管的配子体细胞引导中的作用。同时,该文也对精细胞在花粉管引导中的作用进行了阐述。     

Precocious pollen germination in Arabidopsis plants with altered callose deposition during microsporogenesis

Pollination is essential for seed reproduction and for exchanges of genetic information between individual plants. In angiosperms, mature pollen grains released from dehisced anthers are transferred to the stigma where they become hydrated and begin to germinate. Pollen grains of wild-type Arabidopsis thaliana do not germinate inside the anther under normal growth conditions. We report two Arabidopsis lines that produced pollen grains able to in situ precociously germinate inside the anther. One of them was a callose synthase 9 (cs9) knockout mutant with a T-DNA insertion in the Callose Synthase 9 gene (CalS9). Male gametophytes carrying a cs9 mutant allele were defective and no homozygous progeny could be produced. Heterozygous mutant plants (cs9/+) produced approximately 50% defective pollen grains with an altered male germ unit (MGU) and aberrant callose deposition in bicellular pollen. Bicellular pollen grains germinated precociously inside the anther. Another line, a transgenic plant expressing callose synthase 5 (CalS5) under the CaMV 35S promoter, also contained abnormal callose deposition during microsporogenesis and displaced MGUs in pollen grains. We also observed that precocious pollen germination could be induced in wild-type plants by incubation with medium containing sucrose and calcium ion and by wounding in the anther. These results demonstrate that precocious pollen germination in Arabidopsis could be triggered by a genetic alteration and a physiological condition.     

POLLEN APERTURE POLYMORPHISM AND GAMETOPHYTE PERFORMANCE IN VIOLA DIVERSIFOLIA

Pollen aperture polymorphism is studied in Viola diversifolia, where all plants produce three- and four-apertured pollen grains. We tested whether there are genetic differences among plants for the proportions of the different pollen morphs, and whether the morphs differ in gametophytic performance. Results show that the more apertures a pollen grain has, the more quickly it germinates but that few-apertured pollen grains have faster growing pollen tubes and longer life expectancies. The proportions of the different pollen morphs, together with pollen tube growth rates, may be inherited traits based on differences among maternal families. These results suggest that the different pollen morphs are favored in different pollination ecology situations. The production of several pollen morphs by the same individual could therefore be evolutionarily advantageous.     

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.     

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.     

Lead Stress Disrupts the Cytoskeleton Organization and Cell Wall Construction During <Emphasis Type="Italic">Picea wilsonii</Emphasis> Pollen Germination and Tube Growth

Lead is a widespread pollutant and has been reported to inhibit pollen tube development, but the mechanism of toxicity involved remains unclear. Here, we report that lead stress significantly prevented Picea wilsonii pollen germination and tube growth and also dramatically altered the tube morphology in a concentration-dependent manner. Fluorescence labeling with JIM 5 (anti-acidic pectin antibody) and Calcofluor white revealed the lead-induced decline of acidic pectin and cellulose, especially in the subapical region. Decolorized aniline blue staining showed the marked accumulation of callose in the apical and subapical regions of lead-treated tubes. Fluorescence labeling with Alexa Fluor 568 phalloidin and anti-tubulin antibody revealed that the distribution of the cytoskeleton in P. wilsonii pollen grains and tubes were developmentally regulated and that lead disturbed the cytoskeleton organization, especially in the shank of the pollen tubes. Taken together, our experiments revealed a link between the dynamics of cytoskeleton organization and the process of P. wilsonii pollen tube development and also indicated that lead disturbed the cytoskeleton assembly and, consequently, cell wall construction. These findings provide new insights into the mechanism of lead toxicity in the tip growth of pollen tubes.     

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.     

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.     

Expression of stylar incompatibility in the Andean clonal tuber crop oca (Oxalis tuberosa Mol., Oxalidaceae)

Long-, mid-, and short-styled clonal accessions of oca (Oxalis tuberosa) were intercrossed in a complete diallelic design. Pollen tube growth in styles was monitored in all diallelic crosses. Pollen fertility was estimated by two tests: staining of pollen grains with aceto-carmine and detection of β-galactosidase activity by the substrate X-Gal. The two methods of pollen fertility estimation were equally useful to detect fertility levels. Pollen originating from short stamens had the highest fertility (85%) and pollen from long stamens had the lowest fertility (70%). Pollen fertility was high throughout, but its degree varied with the stylar morph on which the pollen was formed. Long-styled accessions had the highest rates of fertile pollen. Differences in pollen fertility at different anther levels in the same style morph were also apparent. Pollen grain diameter of the six morph-anther level combinations was inversely correlated with pollen fertility. Pollen grains from long stamens were the largest and pollen grains from short stamens were the smallest. Neither pollen fertility nor pollen grain size had an influence on pollen tube growth in the style or on fruit and seed set. Pollen tubes growing within the styles were inhibited at a different level for each of the 18 cross combinations in the diallel. Although legitimate crosses had greatest pollen tube growth, some of the illegitimate inter- and intramorph crosses had equally high scores. Of all illegitimate crosses, mid-styled seed parents had the lowest level of stylar incompatibility. Fruit and seed set were highly correlated with the extent of pollen tube growth in the style. The number of pollen tubes entering ovules in a flower was in good agreement with the number of seeds produced per fruit. Therefore, it is concluded that stylar incompatibility is the major determinant of limited seed formation in oca even in the most successful legitimate cross combinations. Received: 1 September 1999 / Revision accepted: 4 April 2000     

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.     

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.     

Tomato pollen tube development and carbohydrate fluctuations in the autotrophic phase of growth

Ripe pollen has different soluble and insoluble carbohydrates in variable amounts. Pollen germination and pollen tube growth were studied in a tomato cultivar (Solanum lycopersicum L. cv. Platense) with atypical pollen among tomatoes due to its very low amount or absence of sucrose. In vitro assays were performed using a culture medium without carbohydrates to explore whether there is an autotrophic phase of pollen tube growth, and if there is, describe it, and to analyze the fluctuations of endogenous carbohydrates (soluble carbohydrates, starch, pectins, and callose). Pollen germination was fast (ca. 10 min) and a definite autotrophic phase was observed. Soluble carbohydrates and pectins showed the most substantial changes during this period, even after 10 min. A small amount of callose was observed in the ripe pollen and pollen tubes. Pectins were the most abundant pollen tube wall component. Pollen can be considered starchless; starch was not involved in the autotrophic phase of growth. Other types of substances must be connected with the carbohydrate metabolism, because the fluctuations of the different substances did not follow balanced stoichiometric relationships. Pollen germination and pollen tube elongation was sustained autotrophically, even though sucrose was absent and starch was negligible in pollen grains. The type of pollen reserves and the fast pollen tube formation could be selective advantages in this cultivar.     

从柱头到胚囊——花粉管发育研究进展

花粉管的生长和发育是植物有性生殖过程中重要环节。本文首先介绍了花粉管的结构及其脉冲生长方式。然后介绍了花粉粒在柱头上萌发、花粉管在花柱道中生长及其进入胚囊的一些特点,同时还介绍了雌蕊在花粉管生长发育所起的重要作用。     

Kanamycin resistance of germinating pollen of transgenic plants

The influence of kanamycin on the percentage of pollen germination and on tube growth of pollen from non-transformed and transformed plants of various species containing a chimaeric kanamycin resistance gene (NPTII) was investigated. Pollen grains isolated from kanamycin resistant plants expressed resistance when germinating in vitro, whereas kanamycin impaired tube growth of pollen from non-transformed plants. Pollen grains from transgenic plants were less sensitive and produced significantly longer tubes. mRNAs of the chimaeric gene are probably presynthesized concurrently with the other mRNAs during microsporogenesis, and kanamycin resistance is expressed by mRNA translation during pollen tube elongation. Received: 24 August 1999 / Revision accepted: 20 October 1999     

不同倍性香石竹杂交花粉管生长荧光显微观察及结实研究

以香石竹四倍体材料‘紫蝴蝶’(2n=4x=60)为母本,二倍体材料‘珍珠粉’和‘NH6’(2n=2x=30)为父本,利用荧光显微镜观察其授粉后花粉管生长情况,统计其座果率、亲和指数及种子萌发率,并对杂交后代进行倍性鉴定。结果表明,在‘紫蝴蝶’柱头上,‘珍珠粉’和‘NH6’的花粉2h开始萌发,花粉管多处出现胼胝质塞,且花柱组织出现胼胝质反应,4h花粉管到达柱头中部并出现胼胝质塞,6h花粉管到达柱头基部,17h柱头基部的花粉管增多,花粉管进入子房组织且子房组织出现胼胝质反应,17～24h花粉管能与胚珠结合,但结合率低;‘紫蝴蝶’ב珍珠粉’杂交未获得植株,‘紫蝴蝶’בNH6’杂交获得3株植株,染色体倍性鉴定表明3株植株均为四倍体,这可能是‘NH6’产生2n配子的缘故。     

The Pollen Tube Pathway in the Pistil of Lycopersicon peruvianum

The pollen tube pathway has been studied in unpollinated andpollinated pistils of Lycopersicon peruvianum using histochemicalstains for detection of proteins, lipids and arabinogalactansby bright-field microscopy, and decolourized aniline blue fordetection of pollen tubes by epifluoresence microscopy. Thepollen tube pathway is a continuous tract of mucilage from thestigma surface to the ovule micropyles, and is associated witha continuous tract of specialized, protein-rich transmittingcells comprising the stigmatic papillae, vertical files of stylartransmitting cells and the placental epithelium within the ovary.The superficial exudate of the stigma is hydrophobic and richin lipids. The mucilage of the style and ovary is hydrophilicand rich in arabinogalactans but low in proteins. Pollen tubesgrow between cells through the mucilage of the stigma and stylartransmitting tract, and across the surface of the placenta inthe ovarian mucilage. The structure of the junction of the stylartransmitting tract with the top of the ovary placenta assistseffective distribution of pollen tubes within the ovary. Lycopersicon, solanaceae, fertilization, pistil, pollen tube, pollination     

Observation of Pollen Tube Behaviour and Early Embryogenesis Following Interspecies Pollination Between Actinidia deliciosa and A. arguta

The pollen tube behaviour in the style and early embryogenesis following interspecies pollination between Actinidia deliciosa No. 26 and A. arguta were observed by means of fluorescence and light microscopy. Pollen grains germinated on the papillate stigma and pollen tubes grew along the V-shaped open-type style. Pollen tubes showed slower growth and reached the ovules 50--60 hours later than those of the control. Several abnormalities of pollen tubes have been observed at the base of the style, including wave-like pollen tubes, pollen tubes with swollen or pointed tips, with variable diameters, and a few with irregular growth. Random deposition of callose along pollen tube wall and even the whole wall was observed. About 26.74 % of the ovules were successfully fertilized and developed into seeds, among them 68.50% of the seeds were normal and 31.50% were abortive. About 11.41% were empty seeds without embryo and endosperm. Unfertilized small ovule was 61.45 %. Normal seed and its embryo were smaller than those of the control. The development of embryo was of the Soland type. The endosperm was cellular. The zygote remained quiescent for about 12-15 days before it started to divide, eventually forming a cotyledonary embryo 50 days after pollination.