芸苔属脱外壁花粉作为研究花粉萌发的新实验系统

脱外壁花粉的分离与人工萌发已在芸苔属(Brassica L.)中取得成功。由于其缺乏外壁与萌发沟这一特点,脱外壁花粉是研究花粉萌发的有用的实验系统。作者重点研究了脱外壁花粉分离与萌发过程中有关极性形成、萌发位点预定及新壁的合成等问题。结果表明在外壁脱离之前花粉已经活化并预先建立了极性;脱外壁后的萌发位点仍位于原萌发沟处;在萌发位点处合成的新壁可能起限定花粉管直径的作用。由此推论,脱外壁花粉可在花粉生物学研究中广泛加以应用。     

桔梗花粉萌发与花粉管生长研究

以2年生桔梗植株为材料,采用液体培养法研究了培养基种类、PEG、蔗糖、pH以及培养温度、培养时间对桔梗花粉离体萌发生长的影响,结果表明:(1)浓度为100~150 g.L-1的PEG可显著促进桔梗花粉萌发和花粉管的生长;200~250 g.L-1PEG显著促进花粉萌发,但对花粉管生长的作用不显著。(2)100 g.L-1的蔗糖有利于花粉萌发和花粉管生长,高浓度蔗糖(200 g.L-1)有明显抑制作用;(3)桔梗花粉离体萌发和花粉管生长的适宜培养基为ME3+BK+10%蔗糖+150 g.L-1PEG(pH5.8);(4)25~40℃条件下桔梗花粉均可较好萌发,以30℃培养1.5 h为最佳培养条件。     

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。     

云南含笑花粉萌发研究

应用离体培养和人工授粉的方法对云南含笑花粉的萌发进行了研究。栽培和野生云南含笑花粉的萌发率有很大的差异,分别为20％和90％。首次报道了云南含笑的花粉粒在离体培养基上萌发2条花粉管的现象。云南含笑花粉在灰岩含笑花柱头上的萌发率和萌发时间与离体培养基上的相同,表明灰岩含笑的柱头对云南含笑的花粉没有排异现象。     

芸苔属脱外壁花粉的分离与人工萌发

芸苔属青菜（Ｂｒａｓｓｉｃａ ｃｈｉｎｅｎｓｉｓ）与紫菜苔（Ｂ． ｃａｍ ｐｅｓｔｒｉｓｖａｒ． ｐｕｒｐｕｒｅａ）的花粉经低温水合、热激、渗激三步程序,分离出大量具萌发能力的脱外壁花粉,脱外壁率可高达６０％ 以上。在含有碳源与氮源及Ｒｏｂｅｒｔｓ培养基盐成分的碱性ＰＥＧ 培养基中,首次使芸苔属脱外壁花粉萌发,萌发率可达３３％ ～４１％ 。在扫描电镜下观察了花粉脱外壁与萌发的过程。讨论了不同植物花粉脱外壁的方法与花粉壁生物学特点的对应关系,以及外壁对花粉萌发的可能作用     

凯特杏花粉的离体培养及影响因子分析

采用离体培养法,在不同培养基组分含量、pH值、温度及植物生长调节物质的培养条件下,对凯特杏(Prunus armeniacaL.cv Katy)花粉的离体萌发和花粉管生长状况进行观察研究。结果表明:(1)凯特杏花粉离体萌发及花粉管生长的适宜培养基为20%蔗糖 0.04%硼酸 0.01?Cl2,最适pH为6.0,最适温度为20℃,培养20 h后,花粉的萌发率达68.76%,花粉管长度达1 083.53μm。(2)不同植物生长调节物质对花粉萌发和花粉管生长作用不同,赤霉素浓度为5~8 mg/L、矮壮素浓度为10~150 mg/L、多效唑浓度为5~10 mg/L时对凯特杏花粉萌发和花粉管生长都有促进作用,但国光丁酰肼对凯特杏花粉萌发和花粉管生长均有抑制作用。     

杜仲花粉离体萌发特征及花粉管微丝骨架分布

以干燥的杜仲成熟花粉为材料,对杜仲花粉离体萌发的适宜液体培养基配方进行了筛选,并对花粉萌发特征及花粉管微丝骨架分布规律进行了研究。结果表明:(1)适宜杜仲花粉离体萌发的液体培养基组成为200g/L蔗糖+30mg/L硼酸+10mg/L Ca(NO3)2,于26℃条件下离体培养18h的花粉萌发率可达46.29%±3.75%。(2)在适宜液体培养条件下,杜仲花粉萌发率在培养6h内急剧增长,随后趋于平稳;而花粉管在培养8h内伸长较快,之后有放缓趋势,至培养48h时,花粉管长度可达363.14±30.51μm。(3)杜仲花粉属于2胞花粉,花粉萌发过程中,营养核和生殖核的移动存在一定的时序性,通常营养核先于生殖核进入到花粉管;杜仲花粉生殖核的有丝分裂发生在花粉管中,离体培养12h可逐渐观察到有丝分裂行为。(4)花粉萌发过程,微丝骨架形成束状,与花粉管伸长方向平行排布,与较为稀疏的网状微丝阵列组成连续系统,引导细胞核的运动。     

果梅花粉离体萌发及花粉管生长特性研究

研究了果梅(Prunus mumeSieb.1et Zucc.)花粉在不同培养基组分、花粉不同培养密度和不同温度及培养时间的离体萌发和花粉管生长特性.结果表明:细叶青花粉萌发及花粉管生长最适宜的液体培养基为30mmol/L MES(pH 6.5)缓冲液中含20%蔗糖,0.01%硼酸,20%PEG-4000,0.03?(NO3)2?4H2O,0.02%Mg-SO4?7H2O;萌发率达45.03%,花粉管长度达597.2μm.适宜于果梅花粉萌发和花粉管生长的花粉粒密度为20~80粒/μL.培养温度过高或过低都不利于果梅花粉的萌发和生长,25℃时花粉萌发和花粉管生长最好.细叶青"、月世界"、莺宿"3个品种的平均萌发率为48.6%,平均花粉管长度为762.3μm.果梅花粉在不同培养温度下,萌发及生长不同,在25℃条件下花粉管生长速度最快,集中在0~12 h内,3个品种花粉管平均生长速度为58.5μm/h.     

桃花粉离体萌发和花粉管生长特性研究

采用花粉离体萌发法研究不同培养基组分和培养条件对桃花粉萌发和花粉管生长的影响,同时对不同贮藏温度下的桃花粉寿命进行研究.结果表明:固体培养基与液体培养基对桃的花粉萌发率和花粉管长度影响差异不显著;10%蔗糖是大多数桃品种花粉的最适萌发条件;硼能提高桃花粉的萌发率,但对花粉管的生长没有促进作用;桃花粉在20℃～25℃的培养温度下萌发率最高,花粉管最长;桃花粉萌发率和花粉管长度在培养前3 h内上升最快,3～5 h上升趋势减弱,5 h后基本停止;随着贮藏温度的升高和贮藏时间的延长,花粉生活力呈降低的趋势.     

桔梗开花期花粉活力变化对其结实能力的影响

对桔梗花粉离体萌发培养条件进行了筛选,并采用花粉离体萌发法测定了桔梗花粉发育过程中其活力的动态变化,采用人工授粉法对开花后不同时间花粉的授粉结实能力进行了研究.结果显示:(1)桔梗花粉离体萌发的最适温度为30℃,培养1.5 h是花粉管测定的最佳时间.桔梗花蕾期和开花当时花粉离体萌发率和花粉管长度均较低,开花后花粉继续发育,活力逐渐提高,到花后6 h其萌发率和花粉管长度均达到最高,分别为92.2%和602.9 μm,之后活力逐渐下降,花后120 h桔梗花粉的萌发率为63.1%,而花粉管长度只有287.0 μm,仅为花后6 h花粉管长度的47.6%,开花120 h(5 d)后花粉活力下降速度明显加快.(2)桔梗开花后当天花粉人工授粉的结果率、结籽率最高,之后随着花后天数的增加,结果率和结籽率均迅速下降,开花后3 d的花粉结籽率降为0,已完全丧失授粉结实能力.研究表明,在田间条件下,桔梗花粉寿命可保持5 d以上,而授粉结实能力只有2 d左右;花粉管生长缓慢、长度变短可能是导致花后3 d花粉丧失授粉结实能力的主要原因.     

Isolation and Artificial Germination of De-exined Pollen in Brassica

In view of the fact that pollen deprived of exine has been shown by electroporation to be a superior receptor of foreign genes in tobacco, the authors developed a technique to isolate and artificially germinate the de-exined pollen in Brassica. Pollen grains of B. chinens/s and B. campestris var. purpurea were first hydrated at 4 ℃ in a medium containing Roberts' salts, 24% sucrose, 7.3% mannitol, 0.05% lactoalbumin hydrolysate (pH 8.5) for 18 h, followed by heat shock at 34 ℃ for 40～60 min and osmotic shock for 20 min subsequently. Through this three-step procedure the exine could dehisce along the germination furrows and completely detached off, resulting in the release of pollen coated with naked intine with an isolation rate as high, as over 60%. The de-exined pollen were germinated in a modified medium containing 15%～20% polyethylene glycol (MW 6000), 5% sucrose, 0.05% lactoalbumin hydrolysate and Roberts' salts (pH 8.5) with a rate of 41% in B. chinensis and 33% in the other species. However, in sharp contrast with the behavior of the intact pollen, the de-exined pollen did not germinate in the carbon and nitrogen sources free media. Scanning electron microscopic and light microscopic observations on the processes of isolation and germination of de-exined pollen were carried out. The phenomenon of exine detachment in a few plant species is reviewed. There might be some corresponding association between pollen wall characteristics and methods of isolation. The de-exined pollen may be a useful experimental system not only for introducing and transferring foreign genes, but also for investigating the role of exine in pollen germination and in sporophytic self-incom- patibility.     

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.     

Fluorescence Microscopic Observations on Actin Filament Distribution in Corn Pollen and Gladiolus Pollen Protoplasts

Actin filament (AF) distribution in Zea mays pollen and Gladiolus gandavensis pollen protoplasts was localized by FITC conjugated phalloidin fluorescence microprobe. The pollen was incubated in Brewbaker and Kwack (BK) medium, and the pollen protoplasts were isolated enzymatically and cultured in K3 medium containing various supplements by a previously reported method. Samples were fixed for 30 min with 1.5% paraformaldehyde dissolved in 0.1 mol/1 phosphate buffer (pH 7), half strength of BK elements, 1 mol/1 EGTA and sucrose, stained for 30–60 min with 1 μg/ml FITC-phalloidin in the buffer solution, and observed by a fluorescence microscopy. In hydrated corn pollen grains, the AFs constituted an irregular network. Prior to germination a part of the pollen grains showed polarized pattern of Afs. At the opposite pole to the germ pore, there was a center from which AF bundles radiated and converged toward the pore, often making a spindle-shaped configuration. In just isolated gladiolus pollen protoplasts, the AFs appeared as irregular fine network. After 4–7h of culture, the AF distribution coincided in some cases with the unevenly regenerated new wall area as exhibited by FITC-phalloidin and Calcofluor White ST double staining, indicating a possible involvement of AF in wall synthesis. After 17–18 h of culture, a part of the pollen protoplasts went on germination. The AFs became polarized in such protoplasts and converged into the tubes produced, and ran longitudinally along the tubes just like in the tubes germinated from pollen grains. However, in ungerminated pollen protoplasts, the AFs behaved abnormalty, showing various irregular arrangements. When protoplasts bursted, the actin aggregates often located at the protrusion site from which the protoplasts would burst, and were discharged into the medium. In neither corn pollen nor gladiolus pollen protoplasts AFs were observed within the generative or sperm cells.     

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.     

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.     

β-Glucuronidase gene and green fluorescent protein gene expression in de-exined pollen of Nicotiana tabacum by microprojectile bombardment

 Gene constructs containing the β-glucuronidase (GUS) gene or green fluorescent protein (GFP) gene under the control of pollen-specific promoter Zm13-260 from maize were introduced by particle bombardment into de-exined pollen of Nicotiana tabacum. The de-exined pollen exhibited transient expression of the GUS or GFP gene as indicated by histochemical and fluorescent assay, respectively. The frequency of de-exined pollen transformation with the GUS or GFP gene was approximately 6 and 3 times higher, respectively, than that of pollen with intact walls, indicating that pollen deprived of the exine barrier responded better to foreign gene transfer than did the original. Cytological observation of GUS-expressing pollen grains showed that introduced gold particles were visible in the cytoplasm and vegetative nucleus as well as in the generative nucleus. GFP-expressing pollen tubes were observed in the style even after pollination. Received: 28 October 1997 / Revision accepted: 13 April 1998     

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.     

沙田柚花柱蛋白对花粉管生长的影响

本文以沙田柚及其授粉树种酸柚为材料,对其花粉在不同培养介质中的萌发率进行测定,筛选了０２～０４ｍｏｌ／Ｌ聚乙二醇－４００（ＰＥＧ－４００）＋００１％硼酸作为花粉萌发介质。用授粉后３ｄ的花柱提取液进行沙田柚、酸柚花粉萌发抑制实验,花柱提取液对自交花粉萌发无抑制作用,明显地抑制花粉管的生长。其中以花柱提取液的３５％饱和度硫酸铵级分对花粉管伸长的抑制最明显。模拟了花粉在雌蕊中的生长方式,进行花粉萌发生长,能收集到较多的花粉管,为研究花粉管的Ｓ－糖蛋白提供了材料。     

Types of pollen dispersal units in orchids,and their consequences for germination and fertilization

The various pollen dispersal units (PDU) found in orchids are discussed together with possible evolutionary trends and the consequences for germination and fertilization. Orchids with monad and tetrad pollen form more complex dispersal units by means of pollenkitt, elastoviscin, a callosic wall, common walls or a combination of these. Evolutionary trends include (1) from pollenkitt to elastoviscin; (2) from monad to tetrads and multiples of tetrads; (3) from partially dehydrated (<30 %) to partially hydrated (>30 %) pollen; and (4) from monad pollen to PDUs with many pollen grains. The biological consequences concern both male and female reproductive systems. Some features of the male side are present in all orchids irrespective of the pollen dispersal unit, whereas other characters are found only in orchids with pollinia; the same applies for the female counterpart. Pollen grains of orchids with pollinia germinate at least 24 h after pollination because the pollen grains/tetrads must swell and make space for the growth of pollen tubes.