Cytokinesis in microspore mother cells of Impatiens sultani

Summary Cytokinesis in Impatiens sultani microspore mother cells is simultaneous. It starts with the formation of small ingrowths of the surrounding callosic wall. Next, an incomplete cell plate is formed by fusion of small dictyosome vesicles. The cell plate consists of a network of anastomosing tubules and sacs. Aggregates of fusing vesicles are associated with bundles of microtubules, which are oriented perpendicular to the plane of the future cell walls. In the sacculate parts of the cell plate, some callose is deposited, while the associated microtubules disappear. The cell walls ultimately develop by enlargement of the previously formed wall ingrowths, which successively incorporate the elements of the cell plate. The enlargement and thickening of the walls is not accompanied by a further fusion and incorporation of dictyosome vesicles.     

Cytoplasmic differentiation during tetrad formation and early microspore development inImpatiens sultani

Summary From early prophase stage until probaculae formation within the tetrad stage considerable cytoplasmic changes occur. The changes merely concern the ribosome population, the plasma matrix and, the endomembrane system formed by endoplasmic reticulum, dictyosomes and dictyosome-vesicles.The ultrastructure and morphology of mitochondria and plastids remain fairly unchanged, apart from the mobilization of starch during primexine formation.During meiotic prophase there is an increase in ribosome number, accompanied by the presence of nucleoloids in the cytoplasm. Simultaneously the electron density of the cytoplasm strongly increases, indicating a fair increase in protein content. Nucleoloids are also observed in the cytoplasm after primexine formation, accompanied by localized accumulation of ribosomes. Up to the individualization of the microspores the dictyosomes are in an inactive state. After that, they become very active, especially during primexine formation when numerous large dictyosome-vesicles are present.The endoplasmic reticulum (ER), initially in a plate-like configuration, disappears from the cytoplasm during primexine formation. Abundant, smooth and tubular ER is present when probaculum formation starts.     

The microtubule cytoskeleton and the rounding of isolated generative cells ofNicotiana tabacum

Summary Upon squashing of the pollen grain, the isolated generative cell ofNicotiana tabacum looses its spindle shape to become spherical; this phenomenon is independent of the sucrose concentration used. The time necessary for this change can vary from 1 min (0% sucrose) to 20 min (30% sucrose). The microtubular cytoskeleton was studied by means of immunofluorescence and electron microscopy. Just after isolation, 5 to 15 clearly visible bundles in microtubules organized in a basket-like structure are present. After 15 min in medium with 15% sucrose, the microtubular cytoskeleton disappears, and a diffusely spread tubulin can be observed. Neither the addition of 10–20 M taxol to the medium, nor the omission of Ca²⁺ to the medium has any effect on the changes in cell shape and loss of microtubular bundles after isolation.Abbreviations GC Generative cell - SC sperm cell - BK Brewbaker and Kwack - CLSM confocal laser scanning fluorescence micros copy     

Peculiarities of cytomixis in pollen mother cells of transgenic tobacco plants (Nicotiana tabacum L.) with mutant phenotype

The frequency characteristics and cytological picture of cytomixis in the course of male meiosis are described in transgenic tobacco plants (Nicotiana tabacum L.) with altered flower morphology and male sterility. Effects of cytomixis on qualitative composition of meiotic products are studied (formation of cytoplasts and polyads). Doubling of the chromosome number was established to increase frequency of cytomixis in the studied plants.     

Ultrastructure of sperm cells and the male germ unit in pollen tubes ofNicotiana tabacum

Summary The structure of sperm cells and their association with the vegetative nucleus in pollen tubes ofNicotiana tabacum grown in styles were observed with the electron microscope, demonstrating the existence of a male germ unit. The two sperm cells are arranged in tandem and are closely associated with the vegetative nucleus, which always takes the lead. The leading sperm cell (SC 1) has a long and narrow cytoplasmic projection which lies within the enclaves of the much lobed vegetative nucleus, thus forming a physical association. The trailing sperm cell (SC 2) and the SC 1 are not only joined by a common transverse cell wall but also are surrounded by a periplasm bounded by the plasma membrane of the sperm cells and that of the vegetative cell, thus forming a structural connection. The sperm cells are elongated, with cytoplasmic projections at the anterior end of the SC 1 and at both ends of the SC 2. The cytoplasm of both sperm cells includes mitochondria, endoplasmic reticulum, dictyosomes, ribosomes, small vacuoles and axially oriented microtubules. No plastids were observed.Abbreviations DAPI 4,6-diamino-2-phenylindole - MGU male germ unit - MT microtubule - SC 1 the leading sperm cell physically associated with the vegetative nucleus - SC 2 the trailing sperm cell     

Micromanipulation of male and female gametes ofNicotiana tabacum: I. Isolation of gametes

The isolation of male and female gametes is a precondition for the micromanipulation of flowering plant gametes. To reflect their condition at fertilization, isolated gametes need to be physiologically mature and vigorous. Sperm cells are isolated from pollen tubes grown on cut styles using the in vivo/in vitro technique. Embryo sacs are isolated 2 days after anthesis using brief treatments of minimal concentrations of cell-wall-digesting enzymes on ovules of emasculated flowers. Egg cells are then mechanically separated from the embryo sac, allowing unambiguous identification of cells. Two days is usually the minimum required for the pollen tube to penetrate the ovule and effect fertilization in vivo.     

Immunoelectron microscopy of myosin associated with the generative cells in pollen tubes ofNicotiana tabacum L.

In this study, polyclonal anti-myosin antibodies were used for immunogold labeling of ultrathin sections of pollen tubes ofNicotiana tabacum L. to unravel the ultrastructural localization of myosin associated with the generative cells. Clusters of immunogold particles were consistently found in association with the area of the outer surface of the vegetative cell plasma membrane present around the generative cell. Compared to the generative cell cytoplasm, the nucleoplasm showed higher numbers of gold particles. This is the first direct evidence demonstrating the presence of myosin in the nuclei of the generative cell of flowering plants. The possible implications of these findings are discussed in relation to movement of the generative cell in the pollen tube cytoplasm.     

Role of microtubular cytoskeleton and callose walls in the manifestation of cytomixis in pollen mother cells of tobacco Nicotiana tabacum L.

The structure and dynamics of microtubular cytoskeleton and of callose walls in normal pollen mother cells (PMC) of tobacco N. tabacum L. and in cells with intercellular translocation of nuclear material (cytomictic) was studied in the course of the cell cycle. The microtubular cytoskeleton was established as playing no obvious role in the process of cytomixis. The elevated level of cytomictic seems to be due to disturbances of synthesis of callose walls as a result of their attenuation and perforation. Possible causes of cytomictic in tobacco PMC at the cellular level are discussed.     

洋葱花粉母细胞中胞间连丝和胞质通道内的胞质骨架

用DGD包埋去包埋方法,观察了洋葱花粉母细胞中胞间连丝和胞质通道内的胞质骨架分布。结果发现,在花粉母细胞的胞间连丝内有胞质骨加分布,这些骨架纤维集结成束,穿过胞间连丝。在胞质通道内也有胞质骨架分布,但与胸间连丝内的骨困分布有所不同,主要表现为两种形式;骨架纤维致密或稀少。研究讨论了胞质骨架在胞间连丝和胞质通道内的作用。     

An investigation of the role of the anther tapetum during microspore development using genetic cell ablation

The effects on anther development of a fusion of the Arabidopsis anther-specific apg gene promoter to a ribonuclease (barnase) in transgenic tobacco plants were examined. Contrary to expectations, viable pollen grains were produced by these plants despite the demonstration that ribonuclease expression in the microspores and tapetum caused targeted cell ablation. Transformed plants were reduced in male fertility due to ablation of a proportion of pollen dependent on apg-barnase locus number. Plants were otherwise phenotypically normal and fully female fertile, confirming the anther-specific nature of the apg promoter. In microspores inheriting an apg-barnase locus following meiosis, loss of cell viability, as judged by fluorescein diacetate staining, occurred during mid to late microspore development. Microspores not inheriting a transgene went on to mature into viable pollen grains. Premature degeneration of the tapetum was also observed as a result of apg-barnase expression, but this did not appear to disrupt the subsequent microspore and pollen developmental programmes. This was substantiated by observations of microspore development in plants in which the tapetum was rescued from ablation by crossing in a second transgene encoding a tapetum-specific inhibitor of the ribonuclease. It was determined that tapetum cell disruption occurs at the early to mid uninucleate microspore stage in apg-barnase transformants. The data presented show that after this point in microspore development the tapetum is no longer essential for the production of viable pollen in tobacco.     

Enzymatic isolation of viable nucelli at the megaspore mother cell stage and in developing embryo sacs in Nicotiana tabacum

Summary Nucelli and developing embryo sacs were enzymatically isolated from ovules of Nicotiana tabacum. Megaspore mother cells, tetrads, uninucleate, binucleate, four-nucleate, eight-nucleate and mature embryo sacs were obtained. The isolated embryo sacs were intact and living, and maintained their original shape and organization. Cytoplasmic streaming was clearly observed. Prolonged incubation of ovules or reincubation of isolated embryo sacs in the maceration mixture resulted in the liberation of the gametophytic cells as individual, living protoplasts.     

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。     

紫斑牡丹栽培品种小孢子发育过程的细胞遗传学研究

本文对紫斑牡丹栽培品种的花平细胞减数分裂过程进行了系统研究,结果发现紫斑牡丹品种约６８．９６％的花平细胞在减数分裂过程表现正常,约有３１．０４％的花粉母细胞在减数分裂的比线期、中期Ⅰ、后期Ⅰ、中期Ⅱ、后期Ⅱ及四分体时期观察到染色体行为异常。本实验表明,在小孢子形成过程中,多数小孢子发育政党,但有约３１．０４％的花粉母细胞减数分裂异常,导致了花粉的败育。     

A co-culture system leads to the formation of microcalli derived from microspore protoplasts ofBrassica napus L. cv. Topas

Summary This paper describes a procedure in which protoplasts are obtained from microspores and pollen of rapeseed to induce callus formation aided by a feeder cell system with embryogenic microspores. Microspores at late unicellular stage and pollen at early bicellular stage were isolated and precultured for 24 h at 32 °C before enzymatic treatment. Eleven enzymes were tested in various combinations and concentrations. The optimal enzyme combination was 1.0% cellulase, 0.8% pectinase, 0.3% macerozyme, and 0.02% pectolyase, in which 26.3% of the microspores released protoplasts. A successful co-culture system was set up by employing embryogenic microspores as feeder cells. To this end, microspores were cultured in a medium with high osmotic pressure at 32 °C. Up to 37% of the microspores exhibited cell division and embryos developed to the heart-shape stage without changing medium. Microspore protoplasts were cultured in Millicells surrounded by the embryogenic microspores as feeder. In growth-regulator-free medium 14.5% of the protoplasts divided but only formed budding-like multicellular structures. Only after pretreatment with 4 mg of 2,4-dichlorophenoxyacetic acid and 1 mg of naphthaleneacetic acid per liter protoplasts divided and formed microcalli. Pollen tubes or tubelike structures were not observed. The experiments reveal that selection of the specific developmental stage of microspores, which is a prerequisite for microspore embryogenesis, is also important in microspore protoplast culture. Compared to other methods used before, microculture fed with embryogenic microspores has obvious superiority.Abbreviations CPW basic protoplast washing medium according to Power and Chapman - CPW972 CPW basic medium supplemented with 9% mannitol and 7.2% sorbitol - DAPI 4,6-diamidino-2-phenylindole - NLN nutrient medium according to Lichter modified by Pechan and Keller - NLN13 NLN medium supplemented with 13% sucrose - NLNP NLN13 supplemented with 7.2% sorbitol     

太白红杉小孢子的发生和雄配子体的发育

太白红杉(Larix chinensis Beissn)的雄球花7月初开始分化。小孢子囊壁一般包括5～6层细胞：表皮、药室内壁、2～3层中层和绒毡层。绒毡层属于周原质团型。造孢细胞在7月下旬形成,8月上旬形成小孢子母细胞,8月下旬开始减数分裂,于10月上旬进入双线期,并以双线期渡过休眠。翌年3月下旬解除休眠继续进行减数分裂,4月中旬形成四分体,4月下旬到5月初小孢子从四分体内释放出来,小孢子经过连续4次有丝分裂后,于5月中旬形成5-细胞型的成熟花粉粒(雄配子体)并开始散粉。小孢子母细胞发育表现出不同步现象,部分小孢子母细胞在发育过程中出现退化,在小孢子囊内形成空腔。     

Pattern of the plastid division in spore mother cells of the hornwortAnthoceros punctatus

Relative changes in plastid DNA content in each stage of plastid division were investigated in order to better understand the division cycle of plastids in spore mother cells in the horwortAnthoceros punctatus. Samples of cells stained with DAPI were observed with epifluorescence microscopy and CHIAS. In spore mother cells of this species, plastids duplicated their own DNA prior to the plastidkinesis of the first plastid division, but did not replicate plastid DNA prior to the plastidkinesis of the second plastid division. Therefore, the DNA content of those plastids in which division had been completed was reduced to half its initial value. This indicates that the DNA replication pattern of plastids in spore mother cells corresponds to that of cell nuclei during premeiosis and meiosis inA. punctatus.     

A new fluorescent staining method for callose of microspore mother cells during meiosis

To observe the dynamic behavior of callose of microspore mother cells during meiosis, we developed a convenient, rapid and efficient staining method using an improved carbol fuchsin/aniline blue solution. The stained microspore mother cells during meiosis showed yellowish green callose, red cytoplasm and dark red chromosomes when excited with blue light, which produced a contrasting image with a three-dimensional effect. When stained with only improved carbol fuchsin solution, the cells had red cytoplasm and chromosomes when excited with green light. The improved carbol fuchsin solution can be used to replace other more expensive DNA-specific dyes, such as DAPI and H33258, to reduce experimental costs.     

Subcellular localization and topology of homogalacturonan methyltransferase in suspension-cultured Nicotiana tabacum cells

Pectin is a complex polysaccharide in the primary walls of all plant cells that is thought to be synthesized in the cellular endomembrane system and inserted into the wall via exocytosis. The most abundant pectic polysaccharide, homogalacturonan, is partially methylesterified within the cell by the pectin methyltransferase homogalacturonan methyltransferase (HGA-MT). The subcellular location of HGA-MT activity was determined in tobacco (Nicotiana tabacum L. cv. Samsun) cell membranes separated on linear sucrose gradients. The activity of HGA-MT and two enzymatic markers of the Golgi apparatus, IDPase and UDPase, were found to be located in the same membrane fraction. No NADH cytochrome c reductase activity, a marker for the endoplasmic reticulum, was detected in the Golgi fraction. Homogalacturonan methyltransferase activity was not reduced by protease treatment of intact membranes or membranes treated with 0.01% Triton X-100. In contrast, HGA-MT activity was reduced by protease treatment of membranes permeabilized with 0.02% Triton X-100. The sensitivity of HGA-MT in detergent-permeabilized membranes, and the lack of inhibition of HGA-MT activity by protease-treatment of intact membranes, provides evidence that the catalytic site of HGA-MT is located on the lumenal side of the Golgi. Received: 2 December 1998 / Accepted: 9 February 1999     

濒危植物水松小孢子发生和雄配子体发育的研究

水松(Glyptostrobus pensilis)是我国特有的单种属孑遗植物,是极度濒危物种,被列为国家一级重点保护植物。为了从生殖生物学方面探讨水松的濒危机制,采用石蜡切片法对水松小孢子发生及雄配子体发育过程进行了系统地观察研究。结果表明:水松雄球花于10月中旬开始分化,11月初小孢子囊壁形成,12月初小孢子母细胞形成,12月中旬减数分裂。翌年1月中旬形成四面体形和左右对称形四分体,1月下旬四分体解体,释放出游离小孢子。2月中旬花粉粒发育成熟,并以二细胞形态进行传粉,散粉期约为2周左右。3月萌发花粉管,3月下旬出现精原细胞、管核和不育核,5月下旬花粉管到达颈卵器顶部,精原细胞分裂成两个精细胞。水松小孢子和雄配子体发育过程中败育和变形现象很少,据此我们认为,水松小孢子发生与雄配子体发育正常,不是致其濒危的主要原因。     

Micromanipulation of male and female gametes ofNicotiana tabacum: II. Preliminary attempts for in vitro fertilization and egg cell culture

This research is part of an attempt to establish an in vitro fertilization system in tobacco to aid in understanding mechanisms of fertilization. Fusions of isolated male and female gametes were induced in a polyethylene glycol solution. Fusion appears similar to that in maize. One nuclear division of both an unfertilized egg cell and a synergid was induced in KM8p medium with 1 mg/l 2,4-dichlorophenoxyacetic acid in a microchamber culture; one cellular division of the egg cell was also induced in the same medium in solid-drop culture. The osmolality of suspension culture feeder cells was critical for the development of these cells. These results indicate that in vitro fertilization is possible in tobacco, which would be the first such system in dicots.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - PEG Polyethylene glycol