Identification and functional analysis of arabinogalactan protein expressed in pear pollen tubes

Arabinogalactan proteins(AGPs)are widely distributed in the plant kingdom and play a vital role during the process of plant sexual reproduction.In this study,we performed a comprehensive identification of the PbrAGPs expressed in pear pollen and further explored their influences on pollen tube growth.Among the 187 PbrAGPs that were found to be expressed in pear pollen tubes,38 PbrAGPs were specifically expressed in pollen according to the RNA-seq data.The PbrAGPs were divided into two groups of highly expressed and specifically expressed in pear pollen.We further tested their expression patterns using RT-PCR and RT-qPCR.Most of the PbrAGPs were expressed in multiple tissues and their expression levels were consistent with reads per kilobase per million map reads(RPKM)values during pollen tube growth,implying that PbrAGPs might be involved in the regulation of pear pollen tube growth.We also constructed phylogenetic trees to identify the functional genes in pear pollen tube growth.Therefore,19 PbrAGPs(PbrAGP1 to PbrAGP19)were selected to test their influences on pollen tube growth.Recombinant proteins of the 19 PbrAGP-His were purified and used to treat pear pollen,and 11 of the PbrAGP-His recombinant proteins could promote pear pollen tube growth.Additionally,pollen tube growth was inhibited when the expression levels of PbrAGP1 and PbrAGP5 were knocked down using an antisense oligonucleotide assay.PbrAGP1 and PbrAGP5 were localized in the plasma membrane and might not alter the distribution of pectin in the pollen tube.In summary,this study identified the PbrAGPs expressed in pear pollen and lays the foundation for further exploring their functions in pollen tube growth.

Introduction of herbicide resistant gene (bar) into maize (Zea mays L.) via pollen tube pathway

The pollen tube pathway method of transformation has been reported to be successful in most crops, but less successfu in maizc. DNA can be transferred by cutting the stigma following pollination and applying the DNA solution in a suitable period DNA presumably reaches the ovary by flowing down the pollen tube and then integrates into the just fertilized but undivided zygotic cells. To provide the molecular evidence for this procedure, the plasmids pGBIRC carrying a CaMV35S promoter-PPT acetyle transferase （bar） gene-nos terminator genc fusion construct were used. Total 3 276 seeds were produced from the ears trcated with DNA. It was found that 35 scedlings were GUS assay positive, but less intense than that of the positive controls, of which 17 were PCR amplification positive. But, only 13 of the seeds from the plants treated with DNA containing the bar gene were found to be resistant compared with the negative control. Less than 1.07% of progeny seedlings tested cxpressed a herbicide positive reaction and polymerase chain reaction （PCR） with seedling DNA did detect the bar genc. Morphological variation was observed in six plants. We succeed in obtain PPT-resistant maize inbred lines via pollen tube pathway

Pollen Grain Germination and Pollen Tube Growth in Pistil of Rice

The germination of pollen grain in vitro and the growth of pollen tube in the pistil of rice were observed with a microscope. The stigma was removed at different time points after pollination to study its effect on seed setting rate. The rice pollen grain started to germinate at 2 min after pollination and the pollen tube penetrated stigma into style in 5-10 min, 30 min later the end of pollen tube reached the bottom of ovary, and only some pollen tubes arrived at embryo sac at 40 min after pollination. Meanwhile, a small amount of callose began to deposit in the pollen tubes, a great deal of callose was observed at 50 min after pollination, whereas the pollen grain began to shrink. The growing rates of pollen tube in the rice stigma, style and ovary were 1500, 5000, and 5400 μm/h, respectively. The seed setting rate was quite low when the stigma was removed at about 10-15 min after pollination, gradually increased when it removed at 20 min to 50 min after pollination, and over 60% when it removed at 50 min after pollination and finally tended to be stable.

Effects of Ion Implantation on in Vitro Pollen Germination and Cellular Organization of Pollen Tube in Pinus thunbergii Parl. (Japanese Black Pine)

Low-energy ion implantation, as a new technology to produce mutation in plant breeding, has been widely applied in agriculture in China. But so far there is a little understanding of the underlying mechanisms responsible for its biological effects at the cellular level. Here we report the biological effects of a nitrogen ion beams of 30 keV on the pollen grains of Pinus thunbergii Parl. In general, ion implantation inhibited pollen germination. The dose-response curve presented a particular saddle-like pattern. Ion implantation also changed the dimension of the elongated tubes and significantly induced tip swelling. Confocal microscopy indicated that the pollen tube tips in P. thunbergii contained an enriched network of microtubules. Ion implantation led to the disruption of microtubules especially in swollen tips. Treatment with colchicine demonstrated that tip swelling was caused by the disruption of microtubules in the tip, indicating a unique role for microtubules in maintaining the tip integrality of the pollen tube in conifer. Our results suggest that ion implantation induce the disruption of microtubule organization in pollen and pollen tubes and subsequently cause morphological abnormalities in the pollen tubes. This study may provide a clue for further investigation on the interaction between low-energy ion beams and pollen tube growth.

Ion Implantation Hampers Pollen Tube Growth and Disrupts Actin Cytoskeleton Organization in Pollen Tubes of Pinus thunbergii

Pollen grains of Pinus thunbergii Parl. （Japanese black pine） were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin cytoskeleton in the pollen tube cell were investigated using a confocal laser scanning microscope after fluorescence labeling. Treatment with ion implantation significantly blocked pollen tube growth. Confocal microscopy showed that ion implantation disrupted actin filament cytoskeleton organization in the pollen tube. It was found that there was a distinct correlation between the inhibition of pollen tube growth and the disruption of actin cytoskeleton organization, indicating that an intact actin cytoskeleton is essential for continuous pollen tube elongation in Pinus thunbergii. Although the detailed mechanism for the ion-implantation-induced bioeffect still remains to be elucidated, the present study assumes that the cytoskeleton system in pollen grains may provide a key target in response to ion beam implantation and is involved in mediating certain subsequent cytological changes.

Screening of Transgenic Soybean Transformed by Means of Pollen-tube Using Kanamycin

Kanamycin was used to screen To seeds of the variety Dongnong 46 transformed by means of pollen-tube method. The results showed that 400 mg·L^-1 kanamycin could inhibit growth of non-transgenic plants, and 2 positive plants were gotten combined with Gus dyeing and PCR detection. It is proved that this method is economic and effective in preliminary screening the transgenic plants.

Inositol (1,4,5)-triphosphate Modulates Pollen Tube Polar Growth

In this article, the functions of D-myo-inositol-1,4,5-trisphosphate (Ins [1,4,5] P3) in regulating pollen tube polar growth were investigated by application of caged version of the phosphoinositides. To increase the intracellular Ins（1,4,5）P3 concentration at the apical region of pollen tube, the caged Ins（1,4,5）P3 loaded by osmotic shock was activated by 10 s 360 nm UV flash at this domain （10 μm from the tip）. More than 70% pollen tubes were induced swelling at apex and/or growth axis reorientation, accompanying temporarily growth arrest, by localized increase of Ins（1,4,5）P3 concentration （n=21）. While, pollen tubes without being loaded with caged Ins（1,4,5）P3 had not response to the same dosage UV flash. With FM 1-43 fluorescent staining, it was found that growth perturbation by UV activated caged Ins（1,4,5）P3 had tight link with membrane trafficking at the apical zone of pollen tubes. Upon UV pulse, the apical V-shaped bright area where was full of secretory vesicles spread to a much broader region, which implied that actin filaments at the apical region were remodeled. It was also observed that the FM 1-43 fluorescence intensity at tip remarkably increased than that before UV flash, which demonstrated that more secretory vesicles were accumulated at this region. To estimate the role of Ins（1,4,5）P3 in modulating intracellular calcium concentration and distribution, dextran conjugated fluorescent dye Calcium Green-1 and Rhodamine B were microinjected into pollen tubes together with caged Ins（1,4,5）P3. The results showed that calcium concentration at the subapical region increased upon UV released Ins（1,4,5）P3. Consequently, the tip-focused calcium gradient under the apical dome of pollen tube was disturbed. Simultaneously, the pollen tube bulged at the apical region and its growth rate decreased. As the tip-focused calcium gradient was reestablished, the pollen tube morphology and growth recovered to the normal level. Therefore, Ins（1,4,5）P3 can modulate pollen tube growth rate and direction through mobilizing intracellular calcium and regulating vesicle trafficking during pollen tube finding its way to the ovary.

In situ Localization and Isolation of Actin Filaments from Pollen Tubes of Amaryllis vittata Ait

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不同培养基组分对“台农一号”杧花粉萌发和花粉管生长特性的影响

为筛选“台农一号”杧花粉体外培养最适培养基和培养时间,探讨不同培养基组分和培养时间对其花粉体外萌发及花粉管生长的影响,以15%蔗糖+25%聚乙二醇-4000+0.015%硼酸+0.03%钙+0.02%镁+0.01%钾为基础培养基,分别设置1、2、3、4、5、6 h培养,不同蔗糖、硼酸、聚乙二醇-4000、钙、镁、钾浓度梯度单因素及蔗糖、硼酸、聚乙二醇-40003个因素正交试验,统计比较不同处理的花粉萌发率和花粉管生长长度。结果表明,培养时间,培养基中蔗糖、聚乙二醇-4000、硼酸对花粉萌发及花粉管生长均有显著影响,而钙、镁、钾影响不显著。培养2~3 h即可统计花粉萌发率及测量花粉管长度。随着培养基中蔗糖、聚乙二醇-4000、硼酸浓度增加,花粉萌发率及花粉管长度均呈现先增加后降低的趋势,适宜浓度的3个组分能够促进花粉萌发及花粉管生长,浓度过高则起抑制作用。3个因素之间存在明显的互作效应,蔗糖与聚乙二醇-4000的互作效应突出。说明培养基中蔗糖、聚乙二醇-4000、硼酸是花粉体外萌发的必需成分,钙、镁、钾为非必需成分。“台农一号”杧花粉体外萌发及花粉管生长的最适培养基为15%蔗糖+20%聚乙二醇-4000+0.015%硼酸+0.03%钙+0.02%镁+0.01%钾,适宜培养时间为3 h,此条件下花粉萌发率达到83.40%。

报春苣苔属植物杂交亲和性分析及杂交障碍

【目的】探究报春苣苔属属内杂交亲和性及杂交障碍的原因。【方法】以4个报春苣苔原生种、2个栽培品种为材料进行常规杂交,通过花粉活力、柱头可授性筛选父母本后开展杂交工作,初步确定杂交组合亲和性关系,并对授粉后的子房发育跟踪观察。【结果】1)6种报春苣苔属植物的花粉活力和柱头可授性强弱与其杂交亲和性反应没有绝对的因果关系。2)16个杂交组合的子房膨大率均在65.00%以上,结实率有明显差别,以牛耳朵Primulina eburnea、尖萼报春苣苔P. pungentisepala、大根报春苣苔P. macrorhiza、蚂蝗七P. fimbrisepala作为母本时,各杂交组合的平均结实率分别达到了75.55%、78.44%、46.67%、57.78%,表现出了较高的亲和性,而以‘北林之春’报春苣苔P.‘Spring of Beilin’、‘四季’报春苣苔P.‘Four Season’为母本的组合,结实率最高也仅有6.67%,亲和性较弱。3)对5个亲和性较弱的杂交组合进行花粉管行为荧光观察,发现均存在一定程度的受精前障碍,表现为花粉管胼胝质反应、花粉管末端膨大、扭曲断裂,但各组合最终均有花粉管伸入胚珠,从到达胚珠的花粉管数量来看,牛耳朵等3个原生种的花粉数量均可满足胚珠受精需要,但结实率仍较低。【结论】报春苣苔属属内杂交具有一定的亲和性,但部分组合未获得种子,并非由受精前的花粉萌发和花粉管生长所限制,可能是由于受精卵或幼胚的后期发育过程中出现代谢紊乱而导致败育。

金柑杂交授粉生物学特性研究

【目的】掌握金柑花粉活力与柱头可授性的变化情况,寻找最佳授粉时期,探明金柑杂交授粉的生物学特性,提高杂交成功率。【方法】对23份金柑材料进行胚型的统计。选择不同胚数材料,采用TTC染色法和联苯胺-过氧化氢法分别对小蕾期、大蕾期、初开期和盛开期的花粉活力和柱头可授性进行研究。对金柑进行去雄不授粉、自交授粉和杂交授粉处理,比较自交和杂交花粉管的生长情况及3种处理下的坐果率。【结果】罗纹种胚数最少,适合作母本,其次是山金柑、金弹和罗浮,而长寿金柑的胚数最多;花粉活力和柱头可授性均在4个时期呈先增后减的趋势,并均在初开期达到了最高活性;金柑杂交花粉管和部分的自交花粉管均进入了子房;除宁波金弹和宁波罗纹CS外,其他5种金柑种质杂交授粉比自交授粉的坐果率更高,并呈极显著性差异;母本的柱头活性越高,坐果率越高,种内杂交坐果率高于种间,并呈极显著性差异;金柑去雄不授粉下仍能得到果实,具有单性结实的特性。【结论】金柑杂交中应选择单胚型母本材料;金柑属于雌雄同熟,采集花粉最佳时期和授粉最佳时期均为初开期;金柑杂交亲和性比自交亲和性更强,有利于金柑的杂交育种。

攸县油茶远缘杂交亲和性的解剖学研究

【目的】为了探究攸县油茶(Camellia yuhsienensis)远缘杂交亲和性的生物学机制。【方法】研究以石果毛蕊山茶[C.mairei var.lapidea(Y.C.Wu)Sealy](SG)、海南油茶‘海油3号’(C.hainanica‘Haiyou 3’)(HN-3)和普通油茶‘德油6号’(C.oleifera‘Deyou 6’)(DY-6)为父本,采用常规授粉和混合授粉方式对攸县油茶(YX)进行自交和杂交授粉。采用流式细胞仪测定亲本倍性,通过离体萌发法测定父本花粉活力,运用显微技术观察花粉萌发及花粉管生长动态以及早期果实和胚珠发育动态,并田间调查各授粉组合的座果率和落果率。【结果】(1)YX为六倍体,SG为二倍体,HN-3为八倍体,DY-6为六倍体。(2)4种油茶花粉具有较强的活力和育性,其中花粉活力最高的是DY-6,为70.13%;花粉活力最低的是YX,为40.68%。(3)自交和杂交所有组合的花粉均能在柱头上正常萌发,且花粉管在授粉后12 h均生长到花柱基部,但自交和YX×SG的花粉管进入子房后生长受阻,无法到达胚珠;YX×HN-3和YX×DY-6的花粉管则在授粉后72h到达胚珠。(4)自交和杂交授粉后100 d内,YX×DY-6的果实横径、纵径最大,YX×SG的果实横径、纵径最小;YX×DY-6的败育胚珠数最少,平均为3.83枚/果;YX×SG的败育胚珠数最多,平均为6.00枚/果。(5)自交和杂交授粉后100 d内,YX×DY-6的座果率最高,为68.0%;YX×SG的座果率最低,为4.0%;YX×HN-3的座果率为16.0%,其落果高峰主要集中在授粉后0~30 d和40~70 d;混合授粉组合YX×SD的座果率为22.0%。【结论】自交和YX×SG的杂交障碍主要为受精前障碍,YX×HN-3的杂交障碍为受精前和受精后障碍;攸县油茶远缘杂交亲和性也与亲本倍性有关,其亲和性大小为YX(六倍体)×DY-6(六倍体)>YX×HN-3(八倍体)>YX×YX>YX×SG(二倍体);混合授粉方式能在一定程度上提高攸县油茶远缘杂交亲和性。研究结果为攸县油茶远缘杂交亲和性机理研究提供了细胞学基础,也为油茶远缘杂交育种工作提供理论参考。

不同授粉方式对富士苹果花粉管萌发及坐果率的影响

为了探讨不同授粉方式对苹果花粉管萌发及坐果率的影响,以“红富士”苹果为试材,对地熊蜂授粉、意大利蜜蜂授粉、凹唇壁蜂授粉、自花授粉、风媒传粉、人工点花授粉和人工喷花授粉这7种授粉方式的效果进行比较。结果表明,苹果自花授粉和风媒传粉效果差,人工喷花授粉、壁蜂授粉、熊蜂授粉苹果花粉管萌发数量分别为95.18、87.88、93.63个/柱头。意大利蜜蜂、凹唇壁蜂、地熊蜂授粉效果好,花序坐果率都达到100%,均能代替人工对苹果授粉。熊蜂授粉中心花坐果率为99.00%,边花坐果率为20.34%,可以提高中心花坐果率、减少边花坐果率,从而节省人工疏花疏果,减轻劳动力和降低生产成本。这一结果可为果农有效使用授粉昆虫提供技术支撑。综合来看,熊蜂授粉是最佳的授粉方式。

铅对‘早酥’梨花粉萌发及花粉管生长的影响

【目的】为揭示重金属污染影响梨花粉管生长发育的特点及作用机理提供一定的理论依据。【方法】利用含有浓度为0 mg/L、0.1 mg/L、1 mg/L、10 mg/L、50 mg/L、100 mg/L Pb的培养基进行梨花粉的培养,观察不同浓度Pb对梨花粉萌发、花粉管长度及细胞壁主要成分的影响。【结果】Pb抑制梨花粉萌发和花粉管生长,且随着浓度升高,抑制作用越强。此外,Pb改变了梨花粉管细胞壁成分的分布情况:在对照组中,梨花粉管上纤维素均匀分布于花粉管壁表面,胼胝质分布向花粉管顶端逐渐减少,酸性果胶均匀分布于花粉管,酯化果胶富集于花粉管顶端与亚顶端;0.1 mg/L的Pb处理没有明显影响花粉管细胞壁成分分布;100 mg/L Pb处理后,纤维素和胼胝质分别积累于花粉管基部与顶端,酸性果胶含量减少,酯化果胶仅在顶端分布。【结论】低浓度Pb对梨花粉萌发和花粉管生长影响不显著,但是100 mg/L的Pb对花粉萌发和花粉管伸长生长有显著的抑制作用,对花粉管细胞壁成分分布影响也较大。

某变径管状零件液压成形加载路径优化研究

针对现有管件液压成形加载路径优化方法实用性差的问题,提出了试错仿真分析与二分法相结合的加载路径优化方法。首先,通过分析变径管的几何数模、管材力学特性及成形工艺,设计了变径管液压成形模具。其次,进行变径管液压成形实验,与仿真结果进行对比,证明了有限元模型及仿真方法的正确性。最后,利用试错仿真分析与二分法相结合的加载路径优化方法,对变径管液压成形进行加载路径优化,得到了最优加载路径,为管材液压成形加载路径优化方法的工程化应用提供参考。

Classification and Analysis of Tube Hydroforming Processes with Respect to Adaptive FEM Simulations

Tube hydroforming process is a relative new process f or production of structural parts of low weight and high rigidity. The successfu lness of the process depends largely on the a proper selection of loading path w hich is axial feeding distance as related to the applied internal pressure. Due to the complicated nature of plastic deformation, a optimum loading path which w ill guarantee good hydroformed parts free of winkle and fracture has often to be determined by finite element analysis. In order to save trials and errors, adap tive FEM simulation method has been developed. To effectively apply the adaptive simulation method, we have to know the applicability of the method. In this pap er, a classification of tube hydroforming (THF) processes based on sensitivity to loading parameters has been suggested. Characteristics of the classification have been analyzed in terms of failure mode, dominant loading parameters and th eir working windows. It was discussed that the so called pressure dominant THF p rocess is the most difficult process for both simulation in FEM analysis and pra ctical operation in real manufacturing situation. To effectively find out the op timum loading path for pressure dominant THF process, adaptive FEM simulation st rategies are mostly needed.

Histological analysis of pollen-pistil interactions in sour passion fruit plants (Passiflora edulis Sims)

The success of sexual plant reproduction is directly influenced by specific interactions between the pollen and pistil.Light,fluorescence and scanning electron microscopy techniques were used to evaluate the steps of pollination in sour passion fruit plants(Passiflora edulis Sims).In the compatible interaction,pollen tubes grow through stigma projections towards the ovary.The pollen grain surface was found to be spheroidal and to consist of heteroreticulate exine with six colpi.Furthermore,analysis in vivo of pollenpistil interactions indicated that stigmas of flowers 24 hours before anthesis are unable to discriminate compatible(genetically unrelated)and incompatible(genetically related)pollen grains.Taken together,these results provide insight into the cellular mechanisms underlying pollination in passion fruit plants.

Viability and Action of CPL Lectin on <i>in Vitro</i>Germinability of Pollen Grains of <i>Malpighia emarginata</i>DC.—(Malpighiaceae)

This study aimed to observe the viability of the pollen grains of Malpighia emarginata DC. (West Indian cherry) and the action of a lectin in their germination. Lectins are proteins that specifically interact with carbohydrates, but don’t modify them and bind with high affinity and specificity, promoting a transfer of information that is clearly central to many cellular processes in living beings. For the viability test was used aniline blue in lactophenol. The in vitro germination test used was the hanging drop method, in control medium containing sucrose, boric acid, calcium nitrate and agar. The experiment dealt with three different treatments in order to pollen germination: growing medium without addition of lectin, with 1.0 μg/ml and with 3.0 μg/ml CPL lectin. This lectin is extracted from seeds of Crotalaria pallida L.-Leguminosae. Data on pollen grains were analyzed by one-way ANOVA, with 95% reliability and comparison of averages by Turkey test at 5% probability. It was found that despite previous high viability of the pollen grains (c. 70%), a germinability rate has been low, yet exceptionally larger in pollen samples placed on a growing medium with addition of 3.0 μg/ml of CPL lectin (24%). There was no significant difference among the pollen grains germinate in medium without lectin and those in medium with the addition of only 1.0 μg/ml. According to data obtained in the treatments, CPL lectin, with concentration of 3.0 μg/ml, influenced the formation of the pollen tube and thus more pollen germinated in Malpighia emarginata.