Isolation and Fusion of Sperm Cells in Several Bicellular Pollen Species

Living sperm cells were isolated in large quantities from the pollen tubes, grown by the in vivo-in vitro technique in 8 bicellular pollen species belonging to 5 families. An “osmotic shook weak enzyme treatment” method could effectively release sperms from pollen tubes and favor sub sequent purification. The viable sperm yields were up to 82.9% in Zephyranthes candida and 78.2% in Hemerocallis minor. Fusions were successfully induced by polyethylene glycol (PEG) according to the "small-scale fusion" procedure in various combinations, viz., between the same sperm cells in 5 species, between sperm cells of Gladiolus gandavensis and Hippeastrum vitta turn, between sperm cells and microspore protoplasts in Hemerocallis minor, and between sperm cells of H. vittatum and microspore protoplasts of Hemerocallis fulva. Test with fluorochrome reaction, more than 85% of the fusion products of sperm cells in Z. candida were viable. The yieid of viable fusion products between sperm cells and microspore protoplasts in Hemerocallis minor was about 75% and half of them could survive after culture for 24h. The induction of fusion between sperm cells and petal protoplasts in G. gandavensis by a combined PEG-dimethyl sulfoxide (DMSO) treatment was investigated in detail. About 90% of the fusion products thus obtamed were viable. Several critical factors affecting the fusion efficiency were studied. These included the ratio of sperm cell number to petal protoplast number in the mixture, concentrations of PEG and DMSO, and duration of incubation in the inducing solution. It appeared that addition of DMSO could significantly increase the fusion frequency, and that there may be a synergistic effect between PEG and DMSO. This is the first attempt to use isolated sperm cells for fusion studies in bicellular pollen species.     

川百合精细胞的超微结构(英文)

川百合与朱顶红花粉管中的生殖细胞分裂行为非常不同。诸如：染色体行为、微管的组织形式和分布、包括着丝点、微管形成的时间,纺锤体的形状及间期周质微管网络在生殖细胞分裂过程中消失与否等。但这两种细胞具有某些共性,包括在有丝分裂前期缺乏早前期带微管（ＰＰＢ）,末期形成细胞板等。这两种植物精细胞的结构应有较大差异。我们曾报道了朱顶红精细胞的超微结构,本文详细从超微结构方面描述了川百合精细胞的特征。川百合花粉管的萌发采用半离体活体培养方式。１１～１８小时后,ＤＮＡ荧光染料Ｈｏｅｃｈｓｔ３３２５８和醋酸地衣红染色检查花粉管中生殖细胞和精细胞发育时期。切取含有分裂的生殖细胞和精细胞的花柱部分,按曾报道的方法固定、包埋、切片、染色及观察。在所有检查的花粉管中,两精子均前后排列（Ｆｉｇ．１～３）,营养核前导并靠近花粉管顶端（Ｆｉｇ．２,３）。Ｈ３３２５８染色可见两精核间以ＤＮＡ联系（Ｆｉｇ．３）。两个新形成的精核彼此分离（Ｆｉｇ．１）,后来又相互靠近,并维持一定距离（Ｆｉｇ．３）。偶尔一对精子与营养核靠近（Ｆｉｇ．２）。两精细胞被一共同的细胞壁连接,他们不仅被自己的质膜也被营养细胞的质膜包围构成周质。周质平坦光滑。共同壁横向     

Distribution of F-actin and Microtubules in Pollen and Pollen Tube of Lilium davidii

F-actin and microtubules are important components of pollen tube, which have very important function in cytoplasm streaming of pollen tube. The authors observed the distribution of Factin and microtubules in the pollen tube of Lilium davidii Duch. by immunofluorescence technique and confocol laser scanning microscopy, through which some new results were obtained. 1. Chemical fixation could preserve F-actin well in pollen tube, so the relation between F-actin and microtubules could be studied by the methods of chemical fixation and fluorescence labelling in pollen tube. 2. F-actin bundles were absent near the pollen tube tip, while microtubules were abundant and web formed in the pollen tube tip. The authors found that the terminal of microtubules was closely associated with the plasma membrane in the pollen tube tip. 3. Only a few F-actin bundles co-exist with the microtubules in the pollen tube of Lilium davidii. The results provided new evidence for the fimction and relationship between F-actin and microtubules in the pollen tube.     

兰州百合小孢子母细胞减数分裂异常现象的观察

对兰州百合(Lilium davidii var.unicolor)小孢子母细胞减数分裂异常进行了研究,发现存在不等二价体、同源染色体早分离、染色体桥、不均等分离、滞后染色体、核外染色体、微核等。分析了这些异常形成的可能机制及对正常小孢子形成的影响。人工花粉萌发实验表明：小孢子母细胞减数分裂异常是导致花粉败育的主要原因。认为兰州百合长期行无性繁殖引起染色体结构变异,导致减数分裂异常。     

Changes of Microtubular Skeleton in the Generative Cell in Pollen Tube During Mitosis of Lilium davidii

Previous observations indicated that division of the generative cell (GC) in some plant genura such as Lilium and Tradescantia is characterized by several unusual features, including persistence of surrounding microtubule (MT) bundles during mitosis, lacking a matephase plate, the cytokinesis is completed with furrow. The authors have further studied the changes of MT organizations and the chromosome (CHs) behavior in the GC during mitosis using electron microscopy and method of tubulin localizations. No MTs in the GC before GC division and during prophase was seen under electron microscopy. However, there was tubulin in the GC with antitubulin staining. During promatephase to matephase, the CHs appeared and arranged in a complexed tangled pattern lengthwise along the cell. Correspond- ingly, transverse pairs of kinetochores were located along the length and depth of the cell. They stacked successively like the rungs of a ladder. In this phase, a large mount of MTs appeared in the GC, which distributed in the cortex of the cell and among the CHs and along the whole length of the CHs. In the beginning, one or two kinetochore pairs changed from transversely to longitudinally situated in each cell. MTs ended on the kinetochore to form kinetochore MTs (KMTs). With the electron microscopy, authors did not find the image of lateral connection between the MTs and the kinetochores as previous reported with immunofluorescent method. As karyokinesis proceeded, more transverse kinetochore pairs gradually became longitudinal, and KMTs gradually increased in number. However, a distinct spindle was not evidenced. During anaphase, CHs seperation started at various positions along the length of the cell. The distribution of MTs in the GC was similar to that of promatephase to matephase. In late anaphase, the CHs segregated as two groups. Most MTs disappeared but only some remained in the polar regions and the interzone. Authors also measured and compared the lengths of the CHs and indirectly identified the existing anaphase B. During late tolephase, the MTs increased in number gradually in the region between the two newly formed sperm nuclei. The region might be the MT interdigitating zone visualized with antitubulin localization. The MTs disappeared after the cell plate (CP) appeared.     

水稻生活精细胞的分离及细胞学观察

Sperm cells of rice ( Oryza sativa L. ) were isolated from mature pollen grains near anthesis using osmotic shock with sucrose solution. They were separated from pollen contaminants by a Percoll gradient centrifugation. Isolated sperm cells were viable as tested with the fluorochromatic reaction. Cytological observations revealed that the two sperm cells just released from a pollen grain were linked together and each had a long tail-like extension. The isolated sperm cells were spherical with irregular surface and most of them separated, but some still remained in pairs that connected by finger-like structure. As observed with TEM, the isolated sperm cell was surrounded by a single plasma membrane and cell wall did not exist. Sperm nucleus was large and euchromatic.     

Mass Isolation and Preservation of Viable Sperm Cells in Brassica campestris var.purpurea

The two-step osmotic shock and grinding methods reported by Yang and Zhou (1989) were modified for isolation of viable sperm cells in large quantities from pollen grains of Brassica campestris var. purpurea. Factors affecting the yield and survival of isolated sperm cells have been investigated. These included physiological status of donor flowers, sucrose concentration used for pollen hydration, basic media, protectants and osmotica supplemented in the medium etc. As a result, two procedures have been developed. For osmotic shock method, pollen grains at the day of anthesis were hydrated in 25% sucrose solution for 30 min and, after centrifugation and removal of the supernatant, the pellet was shocked by a medium containing 12.5% sucrose, 0.1 g/L KNO3, 0.36 g/L CaCl2, 2H2O, 0.3% potassium dextran sulphate (PDS), 0.6% bovine serum albumin (BSA), and 0.3% polyvinylpyrrolidone (PVP). The viable sperm yield was 34%. After removal of pollen wall debris by filtration and centrifugation, the sperm cell-rich pellets were resuspended in a medium containing 20% sucrose, 5% sorbitol, 0.1 g/L KNOs, 0.36g/L CaCl2·2H2O, 0.6% BSA and 0.3% PDS, and preserved at 4℃ for two days. For grinding method, the pollen grains hydrated in 30% sucrose solution for 30 min. were resuspended in a medium containing 20% sucrose, 5% sorbitol, 0.1g/L QNO3, 0.36g/L CaCl2·2H2O, 0.3% PDS, 0.6% BSA, 0.3% PVP and 20 μg/ml fluorescein diacetate, then ground with a glass homogenizer to release the sperm cells. The viable sperm yield was up to 86%. Following filtration and centrifugation for removal of pollen wall debris, the sperm cells were stored at 4℃ in the same medium but without supplementation of PVP. Tested by fluorochromatic reaction, the sperm cells could survive up to one week with a gradual decline of viability. Cytological observations revealed that pairs of ellipsoidal sperm cells just released were linked together; one of the pair had a long tail-like extension which also show fluorochromasia. Soon after, the sperm cells separated and turned to be spherical. The present results open a prospect to use isolated viable sperm cells for further experimental manipulations.     

Cytological Studies of the Cytoplasmic Inheritance in Lilium regale and L. davidii

The distribution and characteristics of plastids and mitochondria in the generative and sperm cells of Lilium regale Wils. and L. davidii Duch. were described. In L. regale there were few plastids and abundant mitochondria in the newly formed generative cell. When the generative cell became free in the vegetative cytoplasm, the plastids degenerated completely within the generative cell. It was further proved by DAPI fluorescent technique that there was no organell DNA in the generative cell within the mature pollen grain or the pollen tube. However, distribution of the plastids was strictly polarizable during the division of the micmspore in L. davidii, resulting the lack of plastids in the newly formed generative cell. Data of RFLP analysis comparable between L. davidii, L. longifiorum and their interspecific hybrid have also proved the plastid inheritance in L. davidii to be of uniparental maternal transmission. Although the mitoehondria were observed both in the generative and sperm cells of L. regale and L. davidii but their DNA was decomposed in the male gametophyte stage. Therefore the mitochondda in the sperm cell could not be transmitted into the offspring. The results provided the detail, cytological evidence that organelles in the microgametophyte are incapable of genetic transmission in the two species of Lilium.     

兰州百合器官离体培养外植体位置效应观察

探讨兰州百合 (Liliumdavidiivar.unicolor)鳞茎鳞片、叶片和根的不同切段的培养效应。结果表明 :其切段不定芽的分化速度和数量是下段 >中段 >上段。芽的诱导和增殖的最适外植体为鳞茎鳞片 ,兰州百合离体培养中鳞片不定芽诱导和快速繁殖的培养基为MS BA2mg L NAA 0 2mg L ,增殖培养基与诱导培养基相同 ,3周左右不定芽开始分化。叶和根不同部位中不定芽的发育能力大体与鳞茎鳞片一致 ,但低于鳞片 ,较适宜的培养基为MS BA2mg L NAA 0 4mg L ,生根培养基为 1 2MS NAA 0 3mg L ,约 15d生根 ,生根率大于95 %。月增殖率为 1∶4 ,整个繁殖周期约需 3个月。     

被子植物生殖细胞与精细胞的分离方法

开花植物精细胞的发育经历一个独特的后减数分裂过程,在此过程中每个花粉母细胞减数分裂的产物——小孢子经不对称有丝分裂产生1个大的营养细胞和1个小的生殖细胞,随后生殖细胞经过正常的有丝分裂产生2个精细胞。近几年,随着高通量组学技术的不断完善,利用组学技术比较分析生殖细胞和精细胞的分子特征、揭示决定精细胞命运与功能以及受精识别的重要分子已成为植物生殖生物学备受关注的课题。开展此项研究的关键是建立能获得大量高纯度的生殖细胞与精细胞分离纯化技术。该文综述了被子植物生殖细胞和精细胞分离方法的主要研究进展,分析了关键方法的特点和要点以及不同方法之间的差异和共性,以期为相关领域的研究人员提供借鉴。     

Immunoaffinity Chromatographic and ELISA determination of Cytokinins in Germinating Pollens in Lilium davidii Dach.

Liliurn davidii Duch. pollen was germinated in PEG-400. A rapid immunoaffinity chromatograpy system with high efficiency and specificity was adopted to isolate and purify trans-zeatin riboside (t-ZR) and isopentenyladenosine (ipA). Cytokinin determination was carried out by a highly sensitive enzyme linked immunosolvent assay (ELISA) for t-ZR and iPA respectively, t-ZR and iPA were present at the level of 10-8 g/g fr. wt in Lilium davidii pollen. After hydration, t-ZR content of pollen decreased slightly, while iPA increased remarkably, and the total amount of t-ZR and iPA underwent almost no change. Pollen tubes grew fast in the first 3 h after germination, and the amount of t-ZR and iPA also increased greatly both in pollen tubes and in germination medium. The increasing trend of t-ZR and iPA and growth rate of pollen tubes was basically synchronous.     

山丹不同居群花粉形态研究

为掌握山丹居群在孢粉学方面的遗传多样性,在扫描电子显微镜下对25个居群的山丹花粉进行了形态观察,结果表明:山丹花粉粒形态为单花粉粒,极面观为椭圆体,具单萌发沟。不同居群的花粉粒在大小、网眼直径、萌发沟宽、P/E值和外壁纹饰等方面存在不同程度的差异,且居群间花粉性状的变异大于居群内的变异,最大变异系数达25.52%。     

百合小鳞茎离体发生过程中内源多胺水平和多胺氧化酶活性的变化

以百合小鳞茎离体发生为实验系统,研究了鳞片(外植体)内多胺和多胺氧化酶活性的变化与小鳞茎发生之间的相关性。随着细胞脱分化、愈伤组织形成、小鳞茎发生等过程,内源腐胺、亚精胺和精胺的含量均失上升后下降,变化曲线为“钟形”,其含量达最高峰的时间为肉眼可见小鳞茎出现之前。多胺氧化酶活性在小鳞茎发生初期逐渐下降,6d后开始上升,并在小鳞茎突起时达最大值。由于内源多胺水平和多胺氧化酶活性的变化与小鳞茎发生有密切关系,可作为这一形态发生过程的生化指示。     

五唇兰精细胞的分离

五唇兰(Doritis pulcherrima)具二胞花粉,授粉后1 d即有花粉开始萌发,授粉后5 d开始有生殖细胞完成有丝分裂形成一对精细胞。通过人工授粉使花粉管在子房内发育,再利用花粉管直接爆破,成功分离出五唇兰的精细胞。成对的2个精细胞在直径大小、荧光强弱上均显示出较大差异,预示2个精细胞具有不同的前途。     

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。     

兰州百合鳞茎发育及低温解除休眠过程中内源激素的变化

以兰州百合为试材,研究了鳞茎发育过程中以及2、6、10℃条件下保湿贮藏101 d内母鳞茎与新鳞茎中内源激素的变化。结果表明：鳞茎发育过程中内源ABA含量以及母鳞茎的GA₃与ZR含量增加,而内源IAA含量以及新鳞茎的GA₃与ZR含量下降。低温贮藏期间,母鳞茎与新鳞茎的GA₃、IAA含量均有升高过程,而ABA含量呈下降趋势;新鳞茎的ZR含量呈下降趋势,母鳞茎的ZR含量也有升高过程。低温处理初期的34 d内,内源激素变化最为显著。不同贮藏温度相比较,ABA含量差异不大,GA₃含量随温度升高而下降。在富含淀粉的新鳞茎中,GA₃和ABA表现出极显著的负相关关系,而在淀粉含量较低的母鳞茎中GA₃和ABA无相关性。通径分析结果表明,母鳞茎与新鳞茎的物质代谢机制不同,母鳞茎的物质变化受内源GA₃的调控,新鳞茎主要是ABA作用的结果。     

Efficacy of four density gradient separation media to remove erythrocytes and nonviable sperm from canine semen

The objective was to compare four commercially available density gradient centrifugation (DGC) media (ISolate [Irvine Scientific; Santa Ana, CA, USA], Percoll [Pharmacia; Uppsala, Sweden], PureCeption [SAGE In-Vitro Fertilization, Inc.; Trumbull, CT, USA], PureSperm 100 [Nidacon International AB; Molndal, Sweden]) for their ability to separate viable, motile sperm from contaminant nonviable (immotile and/or dead) sperm and red blood cells (RBC). Pooled sperm-rich fractions from four healthy dogs were assessed using Spermvison SAR (Minitube of America). For this, 1 mL of the blood/sperm admixture was pipetted over 4 mL of DGC media: 50%/90% ISolate (Irvine Scientific), 45%/90% Percoll (Pharmacia), 40%/80% PureCeption (SAGE In-Vitro Fertilization, Inc.), and 40%/80% PureSperm 100 (Nidacon International AB). After centrifugation, five 1-mL fractions (A, B, C, D, and E) and the sperm pellet (bottom fraction F) were separated. Sperm morphology and red blood cell/sperm ratio (RBC/S) per fraction were determined on stained slides. All DGC media separated RBC from sperm; the highest red blood cell/sperm ratio was present in ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction A (29.4 ± 29.7 and 28.2 ± 20.8, respectively), and in fractions A and B of both PureCeption (SAGE In-Vitro Fertilization, Inc.) (37.0 ± 22.8 and 39.6 ± 24.3, respectively) and PureSperm 100 (Nidacon International AB) (25.2 ± 5.9 and 23.0 ± 3.9, respectively). The fractions with the highest total sperm recovery, motile sperm recovery, as well as overall motility were ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction D (33.9 ± 29.4%; 40.99 ± 27.9%; 71.2 ± 21.8% and 36.4 ± 14.5%; 39.3 ± 15.8%; 88.6 ± 2.3%, respectively), and for PureCeption (SAGE In-Vitro Fertilization, Inc.) and PureSperm 100 (Nidacon International AB), the sperm pellet, fraction F (78.8 ± 28.3%; 88.0 ± 17.4%; 70.2 ± 11.1% and 73.1 ± 21.0%; 75.4 ± 24.6%; 80.6 ± 17.1%, respectively). In the pellet for PureCeption (SAGE In-Vitro Fertilization, Inc.), more sperm and motile sperm were recovered than in ISolate (Irvine Scientific) and Percoll (Pharmacia) fractions D (P < 0.0163). Therefore, DGC media should be considered for canine semen purification when contaminated with blood or when separation of motile versus immotile sperm is needed.     

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.     

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。     

阳春砂花粉的结构和生殖细胞的分离

阳春砂为自交植物,但花的雌蕊高于雄蕊,是研究单子叶植物纲生殖细胞分子生物学的典型材料。为了解决授粉难的问题,该研究采用植物组织化学方法,对阳春砂花粉的发育过程进行观察,以明确其花粉的结构特征;选择渗透压冲击法分离生殖细胞,以探讨阳春砂的授粉技术方法。结果表明:(1)阳春砂花粉发育的特殊性从四分体时期开始,在四分体时期缺少典型的胼胝质壁,而4个小孢子是由多糖性质的细胞壁分隔、包裹着;成熟的阳春砂花粉粒是二胞型花粉,且花粉细胞质中含有丰富的多糖淀粉和脂滴物质;成熟花粉外壁是由多糖物质构成,而非胡萝卜素和类胡萝卜素酯的孢粉素物质构成。(2)阳春砂花粉在2%甘露醇+5%丙酮的爆破液中,爆破率可达10.67%,并释放出花粉内含物,其中包含生殖细胞;在释放出的花粉细胞质中,生殖细胞可完整保留约30min;用显微操作仪将游离的生殖细胞收集成一定数量的群体,置离心管后即可保存在液氮罐中,每天可收集上百个阳春砂生殖细胞。