绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻活体生殖细胞及精细胞的微丝骨架

利用绿色荧光蛋白(GFP)基因结合鼠Talin基因表达技术及水稻(Oryza sativa L.)转基因技术,筛选出表达稳定和具等位基因型的第三代转基因水稻。在其活体花粉的4个发育阶段(Ⅰ．小孢子晚期;Ⅱ．二细胞早期;Ⅲ．二细胞晚期;Ⅳ．三细胞阶段),观察了细胞内微丝骨架的分布和结构形态的变化。发现在这4个花粉发育阶段,花粉内的营养核、生殖核、生殖细胞和精细胞都在不同的发育阶段出现位移。而这些位移与微丝骨架的结构变化和运动有密切关系。在胞质中央的微丝网络以及细胞周质的网络不断变化和互动,导致营养核、生殖核或生殖细胞和精细胞的定向位移。在活体生殖细胞和精细胞内,存有一股与细胞纵轴平行排列的微丝骨架。这些微丝骨架对生殖细胞及精细胞可以提供移动的动力,这对生殖细胞或精细胞在花管内以及胚囊内的运动(包括独自游动)提供了依据。     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻未成熟花粉的微丝骨架

利用绿色荧光蛋白基因结合鼠Talin基因表达技术及水稻转基因技术,在未成熟花粉发育期(即生殖细胞在形成后从靠壁部位移向中央部位的阶段)的水稻(Oryza sativa L.)内发现了一系列前人未曾报道过的微丝骨架的形成和多变过程.在这一发育阶段,未成熟花粉内的生殖细胞呈圆形,中央部位存有一个大液泡,大量微丝在细胞的中央胞质内形成.微丝首先在营养核的核膜表面形成两个集结中心,中心内的微丝呈短粗状.尔后,中心微丝不断延长,最终在细胞中央的胞质内形成一个非常复杂的类似多个纺锤体结合在一起的网络结构.这一网络的中间部位经常包围着营养核和生殖细胞,网络的部分微丝则与存在周缘细胞质(或称周质)的微丝网络形成连接,在连接点部位则形成一些由微丝环状组成的结构.未成熟花粉中央的微丝网络可能与营养核和生殖细胞在未成熟花粉内的运动有密切关系.     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻未成熟花粉的微丝骨架

利用绿色荧光蛋白基因结合鼠Talin基因表达技术及水稻转基因技术,在未成熟花粉发育期（即生殖细胞在形成后从靠壁部位移向中央部位的阶段）的水稻（Oryza sativa L.)内发现了一系列前人未曾报道过的微丝骨架的形成和多变过程。在这一发育阶段,未成熟花粉内的生殖细胞呈圆形,中央部位存有一个大液泡,大量微丝在细胞的中央胞质内形成。微丝首先在营养核的核膜表面形成两个集结中心,中心内的微丝呈短粗状。尔后,中心微丝不断瞎长,最终在细胞中央的胞质内形成一个非常 类似多个纺锤体结合在一起的网络结构。这一网络的中间部位经常包围着营养核和生殖细胞,网络的部分微丝则与存在周缘细胞质（或称周质）的微丝网络形成连接,在连接点部位则形成一些由微丝环状组成的结构。未成熟花粉中央的微丝网络可能与营养核和生殖细胞在未成熟花粉内的运动有密切关系。     

洋葱花粉母细胞细胞内、细胞间微梁骨架的超微结构观察(英文)

以洋葱(AlliumcepaL.)花粉母细胞为材料,采用DGD包埋去包埋原位技术,对花粉母细胞不同发育时期的细胞内、细胞间微梁骨架的超微结构进行了电镜观察。结果发现,花粉母细胞核内存在粗细不等的微梁骨架,与核仁和染色体紧密相连,随着发育的推移,其均一性发生改变。在核周有核纤层样的结构存在,与细胞核和胞质中的微梁骨架紧密相连,到前期结束时解体。洋葱花粉母细胞内具有发达的胞质微梁骨架,这种结构在减数分裂前期Ⅰ变化不明显。在胞间连接(胞间连丝和胞质通道)内,也有精细的微梁骨架分布,并且与两端细胞中的骨架相连。在凝线期的花粉母细胞中观察到细胞融合现象,有胞质或核内微梁骨架与穿壁转移的胞质小球和核小球内骨架相连。此时细胞核偏向一边,但细胞的其余部位仍充满了胞质微梁骨架。初步探讨了核微梁骨架与核仁和染色体之间的关系,核纤层与细胞核之间的关系,以及细胞内、细胞间微梁骨架与细胞融合之间的关系     

洋葱花粉母细胞细胞内、细胞间微染骨架的超微结构观察

以洋葱（Allium cepa L.）花粉母细胞为材料,采用DGD包埋去包埋原位技术,对花粉母细胞不同发育时期的细胞内、细胞间微染骨架的超微结构进行了电镜观察。结果发现,花粉母细胞核内存在的粗细不等的微染骨架,与核仁和染色体紧密相连,随着发育的推移,其均一性发生改变。在核周有核纤层样的结构存在,与细胞核和胞质中的微染骨架紧密相连,到前期结束时解体。洋葱花粉母细胞内具有发达的胞质微染骨架,这种结构在减数分裂前期Ⅰ变化不明显。在胞间连接（胞间连丝和胞质通道）内,也有精细的微染骨架分布,并且与两端细胞中的骨架相连。在凝线期的花粉母细胞中观察到细胞融合现象,有胞质或核内微梁骨架与穿壁转移的胞质小球和核小球内骨架相连。此时细胞核偏向一边,但细胞的基余部位仍充满了胞质微染骨架,初步探讨了核微染骨架与核仁和染色体之间的关系,核纤层与细胞核之间的关系。以及细胞内、细胞间微染骨架与细胞融合之间的关系。     

栽培甜菜花粉发育过程的超微结构

利用透射电镜技术对栽培甜菜(Beta vulgaris)花粉发育过程进行了超微结构观察。结果表明, 在小孢子母细胞减数分裂期间, 细胞内发生了“细胞质改组”, 主要表现在核糖体减少, 质体和线粒体结构发生了规律性变化。末期I 不形成细胞板,而是在2个子核间形成“细胞器带”。“细胞器带”的存在起到类似细胞板的作用, 暂时将细胞质分隔成两部分。四分体呈四面体型, 被胼胝质壁包围。小孢子外壁的沉积始于四分体晚期, 至小孢子晚期外壁已基本发育完全。单核小孢子时期, 细胞核大, 细胞器丰富。二细胞花粉发育主要表现在生殖细胞壁的变化上, 生殖细胞壁上不具有胞间连丝。成熟花粉为三细胞型, 含有1个营养细胞和2个精细胞。精细胞具有短尾突, 无壁, 为裸细胞, 每个精细胞通过2层质膜与营养细胞的细胞质分开。生殖细胞与精细胞里缺乏质体。     

栽培甜菜花粉发育过程的超微结构

利用透射电镜技术对栽培甜菜（Beta vuigaris）花粉发育过程进行了超微结构观察。结果表明,在小孢子母细胞减数分裂期间,细胞内发生了“细胞质改组”,主要表现在核糖体减少,质体和线粒体结构发生了规律性变化。末期1不形成细胞板,而是在2个子核间形成“细胞器带”。“细胞器带”的存在起到类似细胞板的作用,暂时将细胞质分隔成两部分。四分体呈四面体型,被胼胝质壁包围。小孢子外壁的沉积始于四分体晚期,至小孢子晚期外壁已基本发育完全。单核小孢子时期,细胞核大,细胞器丰富。二细胞花粉发育主要表现在生殖细胞壁的变化上,生殖细胞壁上不具有胞间连丝。成熟花粉为三细胞型,含有1个营养细胞和2个精细胞。精细胞具有短尾突,无壁,为裸细胞,每个精细胞通过2层质膜与营养细胞的细胞质分开。生殖细胞与精细胞里缺乏质体。     

蓝猪耳受精过程中中央细胞和初生胚乳细胞中微丝骨架的组装和细胞核的迁移

通过对蓝猪耳(Torenia fournieri)活体胚囊的研究,发现中央细胞和初生胚乳细胞中的微丝骨架在细胞核迁移时发生了显著的变化.授粉前,微丝在中央细胞的周质位置呈现短束状随机分布.开花两天后,它们组装成截然不同的微丝网络,在这个阶段,次生核位于中央细胞中央位置并与短束状的微丝列阵相联系.在授粉发生后不久,分布在珠孔端的微丝发生片断化,此时次生核与卵器相邻.受精后,初生胚乳细胞核从卵细胞处移开, 在初生胚乳细胞中微丝又重组形成清晰的网络结构.用latrunculin A (LAT-A)和细胞松弛素B(cytochalasin B,CB)破坏微丝骨架,得到的试验结果说明,微丝参与了中央细胞中的细胞核迁移运动.数据也表明,在受精过程中,微丝骨架的动力学特性在中央细胞和初生胚乳细胞的胞质重组中起重要作用.     

朱顶红精细胞超微结构观察

精细胞是双受精作用的直接参与者,是生殖生物学中的重点研究对象之一。以往的研究表明,应用连续超薄切片和计算机辅助三维重组技术,结合免疫荧光定位,发现两个精细胞在体积和细胞器含量上存着差异,即精子的二型性,而且与营养细胞核三者构成紧密功能单位却雄性生殖单位（MGU）。微管对精细胞的性状的确定、运动和维持MGU的动态结构稳定具有重要的作用。本文应用透射电镜。详细观察了朱顶红花粉管中细胞的超微结构,并着重微管结构及其分布的观察。朱顶红成熟花粉为两细胞型。成熟花粉于26℃、黑暗条件下,在液体培养基（含10％蔗糖和100ppm硼酸）中培养13－18小时,然后收集花粉管,固定,供电观察并照相。朱顶红成熟花粉培养13小时后,生殖细胞在花粉管中完成核分裂和胞质分裂等两个过程。形成两个精细胞。形成的两个精细胞前后排列,营养核前导并靠近花粉管顶端。领头的精细胞的细胞质以很大的表面与营养核相互融合（图版1－1,2）,有时营养核与两个精细胞彼此穿插、缠绕（图版1－3）。两精细胞之间的壁上具有多胞质通道和含均质电子密度中等的基质（图版II－4）。精细胞质在核与共同壁之间的区域染色较深,经高倍放大,观察到此处含丰富的微管, 自由分布,但以纵向或斜向为主（图版II－5,6）。所有的微管构成松散的桶状网络存在于两精核之间,除此之外,其他区域无微管分布（图版II－7）。培养18小时之后,两精细胞的共同呈网状（图版II－8）,此时微管均成纵向排列,平行于细胞长轴,构成筐状结构包围状精核,但不构成紧密的束状,证明了前人的免疫荧光观察结果。从以上观察结果我们可以得出如下的结论,朱顶红花粉为两细胞型,因此在花粉管中形成的两个精细胞一般前后排列,跟在营养细胞后面,这种线状排列方式在其他植物中也观察到,可能有利于三者作为一个结构功能单位在花粉管中移动和对花粉管狭窄空间的进化适应。MGU形成得较晚,在生殖细胞和营养核进入花粉管后才形成,并一直维持到精核形成,与其他报道不同。精细胞发育过程中,微管的分布方式变化显著。精细胞中微管的分布仅限于共同的细胞壁和靠近茎核之间的区域,总体构成一个松散的桶状结构。精细胞发育后期,微管均成纵向排列,包围着精核,极似生殖细胞筐状的微管结构形式。     

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。     

Male reproductive cell development in Nicotiana tabacum: male germ unit associations and quantitative cytology during sperm maturation

Generative and sperm cells were examined at four stages of development from generative cell formation to sperm cell maturation using serial transmission electron microscopy. The generative cell and vegetative nucleus are associated in a male germ unit association during pollen maturation and tube elongation, except for generative cell mitosis. At late stages of prophase, this association loosens; the generative cell separates from the vegetative nucleus at metaphase. Slender, unbranched, or occasionally branched projections may be found at one or both ends of the generative cell, or they may be single, blunt, and short. Slender projections are rare during anaphase and telophase. The vegetative nucleus moves back into apposition with one sperm cell at the end of mitosis. During the re-establishment of the association, the vegetative nucleus first touches the end of the leading sperm cell and then moves next to the middle of the sperm nucleus. As the sperm cells enter interphase, a conventional association is re-established between one cell and the vegetative nucleus through one or more long and slender cytoplasmic extensions; these associations are maintained throughout later passage in the pollen tube. During maturation, a significant increase occurs in the surface area of the sperm cells (particularly in the sperm cell in association with the vegetative nucleus), and a lesser increase in nuclear volume and surface area. Other sperm cell parameters, including those of heritable organelles, remain unchanged during sperm cell maturation.     

Mitosis and Amitosis of Generative Cell Nuclei in Artificially Germinated Pollen Tubes in Zephyranthes candida(Lindl.)Herb.

This paper deals with the comportmem of the vegetative nucleus and its spatial association with the generative cell and sperm cells in the artificially germinated pollen tubes of Zephyranthes candida (Lindl.) Herb. before and after generative cell mitosis with the use of DNA-specific fluochrome 4′,6-diamidino-2-phenylindole (DAPI). The induction of amitosis and abnormal mitosis of generative cell nuclei by cold-pretreatment of the pollen prior to germination was studied in particular. In normal case, the generative cell, after appressing to the vegetative nucleus for certain time, underwent mitosis to form two sperms, while the vegetative nucleus became markedly elongated, diffused, and exhibited blurring of its fluorescence. After division, a pair of sperms remained shortly in close connexion with the vegetative nucleus. Then the vegetative nucleus returned to its original state. In the pollen tubes germinated from cold-pretreated pollen, amitosis of some generative cell nuclei were frequently observed. Amitosis took place via either equal or unequal division with a mode of constriction. During amitosis, the dynamic change of vegetative nucleus and its intimate association with generative cell afore described did not occur. Sperm nuclei produced from amitosis could farther undergo amitisis resulting in micronnclei. Factors affecting the amitosic rate of generative cells, such as pollen developmental stage, temperature and duration of cold-pretreatment, were studied. Besides amitosis, cold-pretreatment also induced some abnormal mitotic behavior leading to the formation of micronuclei. Based on our observations and previously reported facts in other plant materials, it is inferred that the vegetative nucleus plays an important role in normal mitosis of generative cell and development of sperms.     

Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana

Summary Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultra-structure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyo-somes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.     

Preliminary observations on the formation of the male germ unit in pollen tubes ofCyphomandra betacea sendt.

Summary The structure of the generative cell and its association with the vegetative nucleus in the pollen tube ofCyphomandra betacea Sendt. were observed with the electron microscope. The generative cell, bounded by its own plasma membrane and the inner plasma membrane of the vegetative cell, possesses the cytoplasmic extension which lies within the embayments of a vegetative nucleus. The generative cell contains the normal complement of organelles and, especially, microtubules which cluster into several groups adjacent to the plasma membrane, oriented along the longitudinal axis of the cell. In the pollen tube reaching the lower end of the style aftersemivivo pollination, both of the sperm cells are elongated and polyribosomes and microtubules are the outstanding feature in the cytoplasm. The two sperm cells are connected by a common transverse cell wall, while cytoplasmic channels exist in both the periplasm of the two sperm cells and the transverse wall. The leading sperm cell (S_vn) is closely associated with the vegetative nucleus. Thus the present study demonstrates the existence of the male germ unit in the pollen tube ofC. betacea. The possible cytoplasmic continuity between the sperm cells and between the gametes and vegetative cell is considered.Abbreviations S_vn sperm cell physically associated with the vegetative nucleus - S_ua sperm cell unassociated with the vegetative nucleus - RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum     

The spermatogenous body cell of the coniferPicea abies (Norway spruce) contains actin microfilaments

Summary In conifer pollen, the generative cell divides into a sterile stalk cell and a body cell, which subsequently divides to produce two sperm. InPicea abies (Norway spruce, Pinaceae) this spermatogenous body cell contains actin microfilaments. Microfilament bundles follow the spherical contour of the body cell within the cell cortex, and also traverse the cytoplasm and enmesh amyloplasts and other organelles. In addition, microfilaments are associated with the surface of the body cell nucleus. The sterile stalk cell also contains microfilament bundles in the cytoplasm, around organelles, and along the nuclear surface. Within the pollen grain, microfilament bundles traverse the vegetative-cell cytoplasm and are enriched in a webbed cage which surrounds the body cell. Microfilaments were identified with rhodamine-phalloidin and with indirect immunofluo-rescence using a monoclonal antibody to actin. The majority of evidence in literature suggests that the spermatogenous generative cell in angiosperms does not contain actin microfilaments, so the presence of microfilaments within the spermatogenous body cell inP. abies appears to be a fundamental difference in sexual reproduction between conifers and angiosperms.     

Brassica napus pollen development during generative cell and sperm cell formation

Summary Brassica napus pollen development during the formation of the generative cell and sperm cells is analysed with light and electron microscopy. The generative cell is formed as a small lenticular cell attached to the intine, as a result of the unequal first mitosis. After detaching itself from the intine, the generative cell becomes spherical, and its wall morphology changes. Simultaneously, the vegetative nucleus enlarges, becomes euchromatic and forms a large nucleolus. In addition, the cytoplasm of the vegetative cell develops a complex ultrastructure that is characterized by an extensive RER organized in stacks, numerous dictyosomes and Golgi vesicles and a large quantity of lipid bodies. Microbodies, which are present at the mature stage, are not yet formed. The generative cell undergoes an equal division which results in two spindle-shaped sperm cells. This cell division occurs through the concerted action of cell constriction and cell plate formation. The two sperm cells remain enveloped within one continuous vegetative plasma membrane. One sperm cell becomes anchored onto the vegetative nucleus by a long extension enclosed within a deep invagination of the vegetative nucleus. Plastid inheritance appears to be strictly maternal since the sperm cells do not contain plastids; plastids are excluded from the generative cell even in the first mitosis.     

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in <Emphasis Type="Italic">Alstroemeria aurea</Emphasis>

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry at different developmental stages of pollen tubes cultured in vitro revealed changes in the nuclear phase at 9 and 18 h after culture. Sperm cell formation occurred at 6–9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cells showed a tendency to increase. This association was suggested as the male germ unit (MGU). When sperm cells, vegetative nuclei, and partial MGUs were collected separately from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial MGUs was counted as 2C. Therefore, the second change in the nuclear phase, which results in an increase in 2C nuclei, is possibly related to the formation of MGUs.     

Microtubule organization in sperm cells in the pollen tubes ofBrassica oleracea L.

Summary Microtubules tightly cross-linked into bundles are described in the sperm cells ofBrassica oleracea pollen tubes. The sperm cells are lobed and tailed and the microtubule bundles are often located in these parts of the cells. In the present paper we suggest that the cross-linked microtubule organization could determine an intertubular sliding, probably generating a motility system that propels the sperm cells through the tube.Abbreviations GC generative cell - Mfs microfilaments - Mts microtubules - SC sperm cell - VC vegetative cell - VN vegetative nucleus