烟草精细胞两个群体的分离

高等植物的倾向受精是一个非常吸引人的研究课题,目前对其机理还不清楚.要想探索高等植物倾向受精现象,前提之一是要分离出一定数量的两个精细胞群体作为分子生物学研究方法的材料.以前的研究表明,烟草(Nicotiana tabacum L.)花粉管中的两个精细胞体积差异明显.这种异型性的精细胞可能与倾向受精有关.烟草是二胞型花粉,生殖细胞只在体内生长的花粉管中才分裂形成两个精细胞.用体内/体外技术培养出花粉管后,爆破花粉管即可释放出花粉管内含物,其中包括两个精细胞.用微量酶液可使两个精细胞分开.然后用显微操作器可挑选出两个大小不同、数量上千的精细胞群体.这种单一纯化的精细胞群体为用分子生物学方法区分两个精细胞的DNA和蛋白质差异打下基础.本研究是高等植物的第二例、二胞花粉植物中的第一例分离两个特定精细胞群体的尝试,为构建烟草两个精细胞的cDNA文库创造了条件.     

Culture And Staining of Pollen Grains of Ricinus communis

Germinating and growing pollen grains (male gametophytes) of Ricinus communis L. in liquid culture is achieved as follows: Pollen is collected over a 10-15 min period from mature anther clusters which have been removed from the male flowers and which have been kept at 25° C and 40-60% relative humidity. Samples weighing between 2.5 and 5.0 mg are brought as quickly as possible into a Desicote treated vial containing 17% sucrose and 30 ppm H₃BO₃ in boiled distilled water. The proportion (w/v) of pollen to culture solution should be 1:100. Shed pollen is kept in a humidity chamber whenever it is not being handled. The air in the culture vial is replaced by O₂ at the pressure of 1 atmosphere plus 5 lb and the sealed vials are shaken gently for 8-10 hr while partially immersed in a waterbath kept at 30° C. The pollen is fixed by the addition to the incubation suspension of an absolute alcohol-lactic acid (4:1) fixing fluid. The proportion used is 36 parts of fixing fluid to 1 part of culture solution. The fixed pollen can be stored in the fixative. Smears are prepared by applying single drops of the constantly agitated suspension of fixed pollen to a microscope slide. After each drop has spread out and dried, an additional drop is added until 10-20 have been applied. The preparations are stained by adding a drop of 1% acetic-orcein and are sealed with fingernail lacquer. The method is well adapted to the following types of studies: pollen germination, physiology of pollen tube growth, morphology of the male gametocyte, and physiology and cytology of the generative cell and nucleus.     

Handling Germinated Pollen on Millipore Membrane During the Feulgen and Autoradiographic Procedures

To prevent loss of pollen during the Feulgen's procedure, the pollen was grown on an autoclaved membrane filter (Millipore AA WP 025 00) in contact with a sterilized medium containing agar 0.5-1%, sucrose according to the genus (Malus 0.3-0.5 M; Persica and Tulipa 0.4 M), and H₃BO₃, 0.01%. To fix the germinated pollen of most species, the membrane was placed for 2 hr to overnight at 2-4 C on filter paper wet with the following mixture: OsO₄, 1 gm; CrO₃, 1.66 gm; and distilled water, 233 ml. To fix Persica pollen, 10% of glacial acetic acid had to be added to the fixative. Washing with distilled water and bleaching with a mixture of 3% H₂O₂ and sat. aq. ammonium oxalate, 1:1, were performed also on filter paper. Similarly, the preparation was processed for Feulgen staining by use of pieces of filter paper wet with the required fluids. Hydrolysis preceding the Schiff's reagent was performed at room temperature with 5 N HCl for 18 min. The differentiation after the Schiff's action was with 2% K₂S₂O₅ buffered to pH 2.3 with 9 ml of phosphate buffer (KH₂PO₄, 1.4 gm; conc. HCl, 0.35 ml and distilled water to make 100 ml). The stained pollen was floated off the membrane with a drop of glacial acetic acid to a gelatinized or an albumenized slide, and squashed. When the coverslip is removed the preparation may be either dehydrated and mounted or coated with autoradiographic film.     

烟草精细胞两个群体的分离

高等植物的倾向受精是一个非常吸引人的研究课题,目前对其机理还不清楚。要想探索高等植物倾向受精现象,前提之一是要分离出一定数量的两个精细胞群体作为分子生物学研究方法的材料。以前的研究表明, 烟草(Nicotiana tabacum L.)花粉管中的两个精细胞体积差异明显。这种异型性的精细胞可能与倾向受精有关。烟草是二胞型花粉,生殖细胞只在体内生长的花粉管中才分裂形成两个精细胞。用体内/体外技术培养出花粉管后,爆破花粉管即可释放出花粉管内含物,其中包括两个精细胞。用微量酶液可使两个精细胞分开。然后用显微操作器可挑选出两个大小不同、数量上千的精细胞群体。这种单一纯化的精细胞群体为用分子生物学方法区分两个精细胞的DNA和蛋白质差异打下基础。本研究是高等植物的第二例、二胞花粉植物中的第一例分离两个特定精细胞群体的尝试,为构建烟草两个精细胞的cDNA文库创造了条件。     

Ca2+对韭兰花粉萌发和花粉管生长的影响

采用离体花粉培养技术,研究不同浓度Ca²⁺对韭兰花粉萌发和花粉管生长的影响。结果表明,较低浓度(10^-3 mol/L)的Ca²⁺对花粉萌发具明显地促进作用,并促进花粉管较快伸长;而过高或过低浓度则起不到促进的作用。     

Intrinsic GUS Activity in Various Tissues and During Pollen Development of Nicotiana tabacum

The β-glucuronidase (GUS) gene has been widely used as a reporter gene in the study of plant molecular biology and genetic engineering. One of the major reasons leading to the popularity of GUS-fusion system was the belief that there was no detectable intrinsic GUS activity in plant tissues. However, investigators have been troubled by the "false positive" results or "background" activities when GUS assays were performed. In the present experiment, histochemical observations of intrinsic GUS activity in various tissues and during pollen development of tobacco (Nicotiana tabacurn L. ) was carried out using 5-bromo-4- chloro-3-indolyl-β-D-glucuronic acid (X-gluc) as a substrate for overnight incubation of the treated tissues at 37℃. No detectable intrinsic GUS activity was found in seedling root, stem, leaf, anther wall and stigma of different stages, ovule, as well as isolated generative cell and embryo sac. During pollen development, two peaks of intrinsic GUS activity appeared, one, close to the microspore mitosis and the other from the full maturation of pollen lasting to the post-germination pollen tube stage, no or weak activity was found at other pollen developmental stages. GUS was located in the cytoplasm of the pollen. The pH value of staining solution strongly influenced the experimental results. Blue color was visualized at pH 5, even when 20% methanol or 0.2 mmol/L glucaric acid-l-4-1actone (GAL, a specific GUS inhibitor) were added. At pH 7, no detectable reaction was found at all. The aforementioned results indicate that when using tobacco pollen as the target of GUS gene transformation, the assay should be strictly controlled to neutral condition for avoiding false positive resuits.     

培养基组分及培养条件对蜡梅花粉萌发及花粉管生长的影响

采用液体培养法研究不同培养基组分和培养条件对蜡梅花粉萌发和花粉管生长的影响。结果表明:(1)PEG-4000是蜡梅花粉离体培养所必需的培养基成分,当培养基中无PEG-4000时,花粉不能正常萌发。(2)培养基内低浓度蔗糖对花粉萌发和花粉管的生长无显著影响,但随着蔗糖浓度的升高,则对花粉萌发和花粉管生长表现出强烈的抑制作用,且浓度越高,抑制效应越强。(3)培养基内其它组分分别在一定浓度范围(0～250g/L PEG-4000、0～50mg/L硼酸、0～30mg/L硝酸钙)内对花粉萌发及花粉管生长有促进作用,但超过上述高限值时则起抑制作用。(4)培养基内镁和钾的浓度对花粉萌发及花粉管生长影响不显著。研究表明,蜡梅最适花粉液体培养基组分为250g/L PEG-4000+50mg/L H3BO3+30mg/L Ca(NO3)2.4H2O,且在pH 5.5、温度15℃和600lx的光照培养条件下蜡梅花粉萌发和花粉管生长最佳。     

应用水稻花粉离体萌发体系观察花粉管内微丝分布

采用非固定、DMSO渗透和异硫氰酸标记的鬼笔环肽（FITC—Ph）染色方法,观察水稻花粉离体萌发过程中花粉管内肌动蛋白微丝的形态和分布。结果表明：（1）水稻花粉水合2min后即可萌发,花粉管生长速度在600～1500μm／h之间。（2）水合而未萌发的花粉粒中,大量较短的梭形微丝束构成微丝网络结构,萌发过程中花粉粒内的梭形微丝束松解,部分微丝转移至萌发的花粉管内沿花粉管纵轴呈束状结构;随着花粉管的伸长,微丝束主要分布在花粉管中前端,但在花粉管顶端区域始终未见明显的微丝束。（3）水合后不能正常萌发的花粉粒内肌动蛋白微丝呈弥散不规则分布,在相同萌发时间生长迟缓的花粉管中,微丝束较少,且主要位于花粉管近萌发孔的部位。表明微丝骨架的形态和分布影响水稻花粉管的萌发和生长。     

Palm Chromosome Studies Facilitated by Pollen Culture on a Colchicine-Lactose Medium

This technique is very useful where pollen is readily available and when roots or microspore mother cells are difficult to obtain or to process. It provides a relatively uniform means of studying chromosomes in a great number of species. Pollen is collected from buds at anthesis or the day before and sown on a medium containing H₃BO₃, 100 ppm; colchicine, 0.04%; lactose, 12%; gelatin, 5%; egg albumen, 1 drop in 10 ml. The gametic chromosome complement is studied at mitosis of the generative cell in the pollen tube. In species which have sufficiently large chromosomes it is possible to construct idiograms for comparative studies. All palm species here reported have a haploid complement of n = 18, and the chromosomes range in length from 0.5-3.5 μ.     

Isolation of Pollen Protoplasts in Nicotiana tabacum

A new method for isolation of quantities of mature pollen protoplasts in Nicotiana tabacum has been established. The first step was to germinate mature pollen in Brewbaker and Kwack medium containing 20% sucrose. When most of the pollen grains had just germinated short pollen tubes, they were transferred to an enzymatic solution for the second step. The enzymatic solution contained 1% pectinase, 1% cellulase, 0.5% potassium dextran sulfate, 1 mol/L mannitol, 0.4 mol/L sorbitol in Dx medium with or without 15% Ficoll. The enzymes firstly degraded the pollen tube wall and then the intine. As a result, intact pollen protoplasts were released with the isolation rate up to 50%-70%. Factors affecting pollen protoplast isolation during the germination and maceration of pollen grains were studied. The suceees depended on two key points:pollen germination duration and osmotieum concentration. The optimal germination duration was 30 rain at 30℃. When it was too long, long pollen tubes formed and subsequently, large number of subprotoplasts instead of whole protoplasts were yielded, as the case reported by previous investigators. The optimal concentration of mannitol and sorbitol in enzyme solution was as high as 1.4 mol/L in total. Lowering of the osmoticum concentration resulted in decrease of percentage of pollen protoplasts.     

小果短柱茶花粉离体培养系统的研究

山茶的短柱茶组是优良种质资源,有必要对小果短柱茶(Camellia confusa Chang 1941)的花粉萌发和花粉管生长的生理特性进行研究.本文研究了花粉生活力、培养温度及pH对小果短柱茶花粉萌发和花粉管生长的影响.结果表明:最适离体萌发培养基为5%蔗糖、0.003%的硼酸,0.005%的氯化钙和12%的PEG...     

Use of Vital Dyes in Conjunction with the Semivitro Technic for the Detection of Pollen Tube Sperm Nuclei

The technique we describe here is a modification of that used by Hough et al. (1985), combined with “semivitro” pollen tube observations. With the semivitro technique, pollen tubes grow from the cut ends of pollinated styles (Brewbaker and Majumder 1961). Pollen of Nicotiana alata was presoaked for 15 min in simplified medium (Brewbaker and Kwack 1963) (10% sucrose, 300 ppm Ca(NO₃)₂, 100 ppm H₃BO₃ with the addition of 0.5 mg/ml of Hoechst 33258 stain from Serva Biochemicals, Heidelberg, Control H, purchased June 1983). (For germination of Nicotiana alata pollen in vitro, we use this same solution, except with 12% sucrose). After this prestaining, the pollen suspension was centrifuged for 5 min at 1200 × g, the pellet resuspended in control Brewbaker medium (i.e., no stain), recentrifuged and used to pollinate detached pistils. The pistils were then incubated at 25 C in a water-saturated atmosphere for 20 hr. At this time, the styles were cut just ahead of the front of the growing pollen tubes (Mulcahy and Mulcahy 1985) and the cut stylar ends each dipped in fresh control Brewbaker medium. Twelve to 24 hours later, tubes growing out of the cut styles were viewed by fluorescence microscopy (exciter filter, BG 12 + KV 418, beam splitter, 500 nm, and barrier filter OG 515). A distinct green fluorescence was seen in the generative and vegetative nuclei (Fig. 1).     

Effects of Nifedipine on Pollen Germination,Pollen Tube Growth and Division of Generative Nucleus in Nicotiana tabacum

The deals with the effects of nifedipine (Nif), a Ca2+ channel blocker of rather high specificity, on pollen germination, pollen tube growth and division of generative nucleus (GN) in experimentlly germinated pollen tubes of Nicotiana tabacurn L. Pollen germination was inhibited by the addition of 10-4 mol/L Nif whereas no significant inhibition by 10-7~10-5 mol/L Nif was observed. The effects of Nif on pollen tube growth were related to its concentration and duration of treatment. At the earlier stage, tube growth was promoted at the lower concentrations (10-7~10-5 mol/L), but was significantly inhibited at a concentration of 10 4 mol/L Nif. With increasing time of culture, even the lower concentrations also became harmful; the stronger the concentration, the earlier the transition from promotion to inhibition. Generally, inhibition of tube growth occurred within 24 hours of culture with different extent in various concentrations. Moreover, higher concentrations also tended to disturb tube morphology and cytoplasmic streaming. Nif was observed to perturb GN division at various concentrations, either blocked it completely at 10-4 mol/L, or only delayed it at 10-7~10-5 mol/L. The dynamics of membrane-associated calcium in pollen tubes was tested with chlorotetracycline (CTC). With increasing time of culture and escalating Nif concentration, CTC fluorescence weakened gradually, indicating that the physiological effects of Nif is mediated by its in hibition on Ca2+ channel activities.     

Ca2+、pH在花粉及萌发花粉管生长中的作用研究进展

花粉正常萌发并生长是精细胞顺利到达胚囊并实现受精作用的前提,因而是高等植物有性生殖的一个关键环节。花粉管生长涉及一系列过程,而花粉(或花粉管)内外的Ca^2 和pH的变化与花粉萌发、花粉管生长有着密切的关系。比较详细地论述了Ca^2 和pH在花粉萌发、花粉管生长过程中的分布特点、生理功能及分子机制。     

Culture And Staining of Pollen Grains of Ricinus communis

Germinating and growing pollen grains (male gametophytes) of Ricinus communis L. in liquid culture is achieved as follows: Pollen is collected over a 10-15 min period from mature anther clusters which have been removed from the male flowers and which have been kept at 25° C and 40-60% relative humidity. Samples weighing between 2.5 and 5.0 mg are brought as quickly as possible into a Desicote treated vial containing 17% sucrose and 30 ppm H₃BO₃ in boiled distilled water. The proportion (w/v) of pollen to culture solution should be 1:100. Shed pollen is kept in a humidity chamber whenever it is not being handled. The air in the culture vial is replaced by O₂ at the pressure of 1 atmosphere plus 5 lb and the sealed vials are shaken gently for 8-10 hr while partially immersed in a waterbath kept at 30° C. The pollen is fixed by the addition to the incubation suspension of an absolute alcohol-lactic acid (4:1) fixing fluid. The proportion used is 36 parts of fixing fluid to 1 part of culture solution. The fixed pollen can be stored in the fixative. Smears are prepared by applying single drops of the constantly agitated suspension of fixed pollen to a microscope slide. After each drop has spread out and dried, an additional drop is added until 10-20 have been applied. The preparations are stained by adding a drop of 1% acetic-orcein and are sealed with fingernail lacquer. The method is well adapted to the following types of studies: pollen germination, physiology of pollen tube growth, morphology of the male gametocyte, and physiology and cytology of the generative cell and nucleus.     

蓝猪耳精细胞的分离及两个精细胞群体的收集

蓝猪耳是二细胞型花粉,生殖细胞在花粉管中分裂形成两个精细胞。用体内-体外技术培养出花粉管后,将其置于爆破液中即可释放出花粉管内含物,其中包括两个精细胞和营养细胞。在显微镜下两个精细胞具二型性：体积较大的精细胞与花粉管的营养核相连,体积较小的精细胞只与大精细胞连接。两个精细胞之间的连接比较结实,需用微量酶液将两个精细胞分开。用显微操作仪就可分别挑选出两个精细胞群体,分别有上百个细胞。蓝猪耳精细胞的成功分离为利用蓝猪耳开展离体受精研究打下了良好的基础。这种单一纯化的精细胞群体的获得为用分子生物学方法区分两个精细胞的特异基因和蛋白质创造了条件。     

Pollen germination, tube growth and morphology, and microtubule organization after exposure to benomyl

The inhibitory effects of benomyl on pollen tube growth have received little attention, particularly at the microscopic and immunohistochemical levels. Pollen germination and tube growth in the presence of benomyl were evaluated in Tradescantia virginiana to investigate the effects of this fungicide on pollen germination rate, tube growth and morphology, and microtubule (Mt) organization. Benomyl was incorporated in germination media at 0,480, 600 or 720 mg 1⁻¹. Inhibition of pollen germination, cytoplasmic streaming, and tube elongation were associated with benomyl treatments. Benomyl also induced abnormal pollen tube morphology and Mt organization. Compared to controls, Mts in the treated tubes were characteristically fewer in number, fragmented, sinuous and increasingly disorganized. At the two highest benomyl concentrations, Mts were considerably fewer or absent in apical/subapical regions of the pollen tubes. This work verifies that benomyl incorporated into germination media at concentrations lower than recommended field rates inhibit pollen germination and tube growth, and that the effects are associated with alterations of Mt organization.     

花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响

以烟草为材料,通过半体内实验,就花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响进行了观察。发现用EGTA及钙调素抗血清处理柱头或花粉均可抑制花粉在柱头上的萌发;向花柱引导组织中显微注射纯化钙调素可促进花粉管束伸长,而注射钙调素抗血清可抑制花粉管束伸长;同时证实玉米花柱和花粉细胞壁中均存在钙调素及钙调素结合蛋白,而且花粉和花柱细胞壁中钙调素结合蛋白的种类有差异。结果表明存在于花粉和花柱细胞外的钙调素对花粉萌发和花粉管伸长均有促进作用。     

Subcellular Localization of the S Locus F-box Protein AhSLF-S2 in Pollen and Pollen Tubes of Self-Incompatible Antirrhinum

The distribution of the S locus F-box (SLF) protein was examined by immunocytochemistry and Western blot techniques using an antibody against the C-terminal part of AhSLF-S2 in self-incompatible Iines of Antirrhinum. Abundant gold particles were found where pollen tubes emerge in vitro. With the elongation of pollen tubes, binding sites for the antibody were found in the cytoplasm of the pollen tubes,including the peripheral part of the endoplasmic reticulum. After germination in vitro for 16 h, the product of AhSLF-S2 and possibly its allelic products could still be detectable, implying that the SLF protein has a role in the elongating process of pollen tubes. The present study provides evidence at the protein level that the SLF protein is present in pollen cytoplasm during pollen tube growth. These findings are discussed, as is their potential role in the self-incompatible response in Antirrhinum.     

Staining Germinating Pollen and Pollen Tubes

Germinating pollen on stigmas and pollen tubes in styles of Antirrhinum, Brassica, Oenothera, Raphanus, Rosa, solatium and Tagetes spp. were prepared for examination as follows: The styles were fixed in ethyl alcohol-acetic acid 3:1 for 1 hr, and hydrolyzed at 60°C for 5 to 60 min (depending on the species) in 45% acetic acid. The stigma with its attached strand(s) of stigmatoid tissue was then dissected out under a stereoscopic microscope, placed in a few drops of a staining solution made by dissolving 150 mg of safranin O and 20 mg of aniline blue in 25 ml of hot 45% acetic acid. After 5-15 min in this stain, the tissue was placed in a fresh drop of stain on a microscope slide and gently squashed under a cover glass. Because of a gradual precipitation of the aniline blue component, the stain had to be filtered regularly before use. However, a staining solution could be kept at room temperature for several weeks.