Cytoplasmic DNA apportionment and plastid differentiation during male gametophyte development in Pelargonium zonale

In the male gametophyte of Pelargonium zonale, generative and sperm cells contain cytoplasmic DNA in high density compared to vegetative cells. Cytoplasmic DNA was examined using the DNA fluorochrome DAPI (4'6-diamidino-2-phenylindole) and observed with epifluorescence and electron microscopy. The microspore cell contains a prominent central vacuole before mitosis; mitochondria and plastids are randomly distributed throughout the cytoplasm. Following the first pollen grain mitosis, neither the vegetative cell nor the early generative cell display a distributional difference in cytoplasmic DNA, nor is there in organelle content at this stage. During the maturation of the male gametophyte, however, a significant discrepancy in plastid abundance develops. Plastids in the generative cell return to proplastids and do not contain large starch grains, while those in the vegetative cell develop starch grains and differentiate into large amyloplasts. Plastid nucleoids in generative and sperm cells in a mature male gametophyte are easily discriminated after DAPI staining due to their compactness, while those in vegetative cells stained only weakly. The utility of the hydrophilic, non-autofluorescent resin Technovit 7100 in observing DAPI fluorescence is also demonstrated.     

Ultrastructure of the early stages of carposporophyte development in the red algaChondria tenuissima (Rhodomelaceae,Ceramiales)

The ultrastructure of the early stages of carposporophyte development in the marine red algaChondria tenuissima has been studied. The diploid carposporophyte grows on the gametophyte. Apical gonimoblast cells develop into diploid carpospores. The basal gonimoblast cells cease to divide and undergo considerable cytoplasmic changes before they become incorporated into the expanding fusion cell. Nucleus and plastids degenerate gradually, while mitochondria remain intact. The smooth endoplasmic reticulum becomes prominent, it seems to produce small vesicles with electron dense contents. Simultaneously, numerous mucilage sacs are formed, presumably from dilating ER cisternae. The contents of the mucilage sacs are secreted by exocytosis. The pit connections between gonimoblast cells flare out. They remain as isolated bodies without connection to a wall after fusion. Secondary pit connections occur between vegetative gametophyte cells and sterile carposporophyte cells. There are three different morphological types of pit connections.     

The development of the placenta in the anthocerote Phaeoceros laevis (L.) Prosk

The development of the placenta in the anthocerote Phaeoceros laevis (L.) Prosk. was studied by transmission electron microscopy. By the time the sporophyte emerges from the involucre, a conspicuous placental region is formed by the intrusive growth of sporophyte foot haustorial cells into the adjacent gametophyte vaginula tissue. The separation of gametophyte cells by haustorial cells and their incorporation into the placenta are preceded by the loosening and swelling of their walls and the formation of a periplasmic space. This process causes the disruption of the plasmodesmata, and may eventually result in the complete isolation and consequent degeneration of the cells. Crystals are commonly observed in the vacuoles of gametophyte placental cells. Crystals become more abundant during cytoplasmic degeneration, and are released in the placental lacunae that result from the complete dissolution of gametophyte cells. During the subsequent phase of capsule elongation, the gametophyte placental cells that retain the symplastic connection with the adjoining gametophyte parenchyma develop a wall labyrinth typical of transfer cells. Obliteration of the wall labyrinth by deposition of lightly staining wall material is observed later in sporophyte development, in concomitance with capsule dehiscence. Crystals are negative to the periodic acid/thiocarbohydrazide/silver proteinate test for carbohydrates whilst they are completely digested by pepsin or protease, denoting protein composition.Abbreviation PATAg periodic acid/thiocarbohydrazide/silver proteinate     

云贵水韭雌配子体发育及其濒危机制的研究

为了解云贵水韭(Isoetes yunguiensis)的生殖过程和濒危原因,采用人工培养获得其雌配子体,用半薄切片法观察雌配子体发育过程。结果表明,云贵水韭的大孢子萌发率约为26%;雌配子体异养,终生都在大孢子细胞壁内发育;颈卵器发育只有2枚颈沟细胞而无腹沟细胞。雌配子体发育停止在游离核时期、颈壁细胞数量偏少或排列紊乱、颈沟细胞异常、卵细胞凋亡等可能是导致生殖濒危的原因。同时,游离核是水韭生殖生物学研究和系统学研究的重要环节之一;缺失腹沟细胞可能是水韭类简化性进化的特征之一。     

烟草大小孢子发生和雌雄配子体发育研究

采用卡宝品红染色压片和石蜡切片法对迪勃纳氏烟草(Nicotiana debneyi)大小孢子发生和雌雄配子体发育过程进行了研究.结果发现:(1)迪勃纳氏烟草为两性花,其小孢子发生和雄配子体发育早于大孢子.(2)迪勃纳氏烟草小孢子发生和发育过程基本正常,减数分裂过程有少数细胞出现滞后染色体、染色体断片和染色体桥等异常现象;其胞质分裂为同时型,四分体为十字交叉型和正四面体型,成熟的花粉粒为2细胞型.(3)迪勃纳氏烟草为2室子房,中轴胎座,倒生胚珠,胚珠多数,胚囊发育为蓼型,大孢子发生和发育过程未观察到异常现象.     

槲蕨配子体形态发育的扫描电镜观察

该研究以水龙骨科(Polypodiaceae)槲蕨(Drynaria roosii Nakaike)为研究对象,在人工培养条件下,采用扫描电镜观察其配子体发育的全过程,以期从三维立体角度揭示槲蕨配子体各发育阶段中的一些精细结构,为进一步补充部分经典形态学理论提供依据。结果显示:(1)槲蕨孢子萌发过程中原叶体母细胞的初生假根存在两条同时生长的现象。(2)槲蕨配子体的假根膨大及分叉现象普遍,且具有2层细胞壁,基部呈圆孔状结构。(3)槲蕨的原叶体腹面及边缘处毛状体非常发达,且乳突状单细胞毛状体和针状单细胞毛状体混生。(4)精子器释放精子时,盖细胞前后呈不同开裂状态。(5)发现精细胞向游动精子转化可能的限制性结构是细胞膜,细胞膜表面附着有碎屑状物质。(6)观察到腹沟细胞的解体过程是从近颈卵器开口处的细胞膜开始逐渐向四周及其后下方扩展。(7)发现了颈壁细胞在排列顺序和数量上都不稳定的畸形颈卵器、体积正常但无法区分雌雄的性器及体积显著增大呈锥形的泡状败育性器。     

Stigmoid bodies contain type I receptor proteins SorLA/LR11 and sortilin: new perspectives on their function.

Stigmoid bodies (SBs) are structures in the cytoplasm of neurons. SBs are mostly found in the hypothalamic region of the rat and contain a protein called huntingtin-associated protein 1 (HAP1). In a recent publication, large cytoplasmic structures were shown to be immunoreactive for a type I receptor called SorLA/LR11. By light microscopic analysis, these structures appeared similar to SBs in size and in brain regional and subcellular localization. To determine whether these large puncta correspond to HAP1-containing SBs, we used antibodies specific to various domains of the apolipoprotein receptor LR11 to perform immunocytochemistry in rat and mouse brain tissue. Transfection studies using HeLa cells were conducted to demonstrate the specificity of the antibodies. We found that, in both species, antibodies to the domain II (or VSP10 for vacuolar sorting protein 10 domain) of LR11 immunoreact with large cytoplasmic structures. Co-localization immunolabeling experiments in rat brain tissue sections and in neuron cultures showed that these LR11-immunoreactive structures correspond to HAP1-positive SBs. Electron microscopy was performed in rat hypothalamus and further demonstrated the presence of LR11 in SBs and its co-localization with HAP1. LR11-containing SBs were most abundant in the hypothalamus but were also found in many brainstem nuclei, thalamus, and hippocampus. Our data also show that sortilin, another transmembrane protein containing a VPS10 domain, localizes to large cytoplasmic puncta and is found in LR11-positive and Hap1-positive SBs in hypothalamic neuron cultures.     

Postpollination-prezygotic ovular secretions into the micropylar canal inPseudotsuga menziesii (Pinaceae)

Ovular morphology was examined ultrastructurally inPseudotsuga menziesii to determine the effects of the ovule on pollen development. Vesicles containing lipid-like substances traverse cell walls of the inner epidemis of the integument and release their contents at the integument surface to form the integumentary membrane. A major aqueous secretion from the integument into the micropylar canal is proposed to occur by the movement of the integumentary membrane and its invaginations towards the center of the micropylar canal. The cellular degeneration of the nucellar apex results from the breakdown of vacuoles. After this degeneration, electron-dense substances move from the prothallial cells of the female gametophyte towards the nucellus, and many morphological changes in the nucellus, prothallial cells, and micropylar canal take place simultaneously. We interpret these changes to result from another major secretion from the prothallial cells. Egg cytoplasm appears to disorganize for a short time. Simultaneously, substantial amounts of electron dense-substances in the prothallial cells and lipid-like substances in surface cell walls of the female gametophyte move towards the nucellus as components of the third major secretion.     

Free taxanes and the release of bound compounds having taxane antibody reactivity by xylanase in female,haploid-derived cell suspension cultures ofTaxus brevifolia

Summary Haploid cells derived from the female gametophyte ofTaxus brevifolia produce multinucleate egglike cells that have grown as cell suspensions over a 5-yr period. Two new media are formulated, one without nitrates, to select for haploid cell lines and to enhance free taxane production when cell lines are artificially aged by withholding subculture. The cell suspensions produce bound taxanes that are detectable with anti-taxane monoclonal antibodies on cell surfaces and on particles released into the culture medium. Recovery and detection of bound taxanes are based on a competitive inhibition enzyme immunoassay (CIEIA). Tissues extracted exhaustively with methanol then treated with xylanase release CIEIA-detectable taxanes. Approximately 300% more taxanes were detected in the xylanase-treated tissue culture material versus the control. Wood (winter bark) taken fromT. brevifolia and treated in the same way with xylanase release 20% more taxanes than could be detected in the control.     

THE REGULATION OF ALTERNATION OF GENERATION IN FLOWERING PLANTS

The developmental changes involved in the alternation of generation represent the major gene-switching events in the life history of plants. While a large number of genes are common to both sporophyte and gametophyte, many thousand sequences are specifically expressed in each generation; indeed, certain key constituents (e.g. tubulin) are encoded by different genes in each generation, indicating that sporophyte and gametophyte are responding to different evolutionary pressures. Evidence is accumulating that major gene-switching events in plants, such as flowering, are regulated by complex control systems which ensures that development occurs only in the correct groups of cells at the appropriate time. A similar, or more sophisticated system might thus be expected to regulate alternation of generation. It is not possible to manipulate alternation of generation in a similar fashion to flowering, but study of apparent aberrations of development occurring in nature and in vitro suggests that alternation only occurs in cells which have become competent to receive particular developmental stimuli. Further, in certain cases, competent cells may be switched either into sporophytic or gametophytic developmental pathways depending upon the nature of the stimulus. Acquisition of competence seems to involve isolation of cells from the symplast, some cytoplasmic dedifferentiation, and perhaps cell cycle arrest or transition. The stimuli in vivo appear metabolic in nature, although embryogenesis may be activated by specific classes of glycoproteins. Interestingly, examination of agamospermic systems suggests that fertilization of the egg per se is not the signal which activates sporophytic development. Once competent cells have received the stimulus they start to develop, with no delay in a ‘determined’ state. Sporophytic and gametophytic development in vivo and in vitro both start with an asymmetric division, except for the female gametophyte which may arise via a range of developmental pathways, depending on the species.     

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

Summary In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.     

Tinctorial Properties of Spherical Bodies in Broth Cultures of Nocardia asteroides GUH-2

In yeast extract-supplemented brain heart infusion (BHI) broth cultures of Nocardia asteroides GUH-2, many spherical bodies (SBs) were frequently seen nearby filamentous cells. They showed no Gram-positivity when Gram stain was applied. When acridine orange stain was applied, many of them showed different green fluorescence from bright orange fluorescence of the filamentous nocardiae under ultraviolet light. Their acid-fastness appeared to depend on the presence of paraffin. Using the polymerase chain reaction (PCR) method, 16S rRNA genes were detected in SB-containing broth cultures inoculated with culture filtrates from broth cultures of the strain and identical to that of N. asteroides. These results suggest that SBs are cell wall-defective (CWD) forms which result from the spontaneous mutation of N. asteroides GUH-2.     

孝顺竹(Bambusa multiplex)大孢子发生与雌配子体发育研究

为了解孝顺竹（Bambusa multiplex）的大孢子及雌配子体的发育过程,利用扫描电镜对孝顺竹的雌蕊形态以及大孢子和雌配子体的发育进行了观察。结果表明,孝顺竹雌蕊单子房,1室,双珠被,薄珠心;大孢子母细胞是由1个雌性孢原细胞直接发育而成,大孢子四分体为线性,位于珠孔端的1个大孢子分化成为功能大孢子,然后由功能大孢子依次经历二核、四核、最终形成1卵细胞2助细胞2极核3反足细胞的成熟胚囊。此外,孝顺竹为雌雄同熟类型,根据雌、雄蕊发育的对应关系,从雄蕊形态可估测雌配子体发育阶段。有少数雌蕊出现败育现象,可能是孝顺竹结实率低的原因之一。     

Comparative development of heavily asymmetric-cordate gametophytes of <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> (Anemiaceae) focusing on meristem behavior

Development of heavily asymmetric cordate gametophytes of Anemia phyllitidis (Anemiaceae), one of the schizaeoid ferns, was examined using a sequential observation technique; epi-illuminated light micrographs of the same growing gametophytes were taken approximately every 24 h. The apical cell-like wedge-shaped cell was produced once from the terminal cell of a germ filament, but it stopped dividing soon after production of one or two derivative cells. Without a functional apical cell, the gametophyte developed by intercalary growth until the early stage of wing formation, and then the multicellular (pluricellular) meristem arose from the lower lateral side of the gametophyte. This was in sharp contrast to the observation that the multicellular meristem forms in place of the apical cell in typical cordate gametophytes. Loss of the functional apical cell probably caused a site-shift in the multicellular meristem of the Anemia phyllitidis gametophyte during evolution from apical to lateral. The results suggest that apical cell-based and multicellular meristems are primarily independent of each other. The multicellular meristem produced cells equally in the distal and proximal directions to form wings in both directions but proximally produced cells divided much less frequently. As a result, a heavily asymmetric gametophyte was formed.     

Photolithotrophic cultivation of <Emphasis Type="Italic">Laminaria japonica</Emphasis> gametophyte cells in a silicone tubular membrane-aerated photobioreactor

Gametophyte cells of brown algae Laminaria japonica were employed both in a modified silicone tubular membrane-aerated photobioreactor (bubble-less cultivation mode) and a bubble-column photobioreactor (bubbling cultivation mode), to study different gas–liquid mixing modes on cell growth rate and cell physiological status. With an inoculum density of 50 mg DCW l⁻¹, in modified artificial Pacific seawater (APSW) medium at 13°C, light intensity of 60 μE m⁻² s⁻¹, light cycle of 16/8 h L/D, and aeration rate of 60 ml min⁻¹, the specific growth rates were 0.082 d⁻¹ for bubble-less mode and 0.070 d⁻¹ for bubbling mode with biomass, in the form of dry cell density, increasing 10.9 and 6.8 times, respectively, during the 36 days’ photolithotrophic cultivation. The specific oxygen evolution rate under bubble-less mode was 39.6% higher than under bubbling mode on the 18th day. The gametophyte cells grew in cell aggregates with clump sizes, at day 36, of 1.5 mm and 0.5 mm diameter under bubble-less and bubbling mode respectively and cell injury percentages of 5.1% and 21.1%, respectively. The silicone tubular membrane-aerated photobioreactor was better suited for the cultivation of fragile macroalgal gametophyte cells due to the absence of hydrodynamic shear stress caused by fluid turbulence and the presence of a bubble-less gas supply.     

Characteristics of spore germination and protonemal development in Hypnum pacleseens

The spore germination, protonemal development, and gametophyte differentiation of Hypnum pacleseens were observed in cultivation. Photomicrographs showed that spore germination of Hypnum pacleseens occured within the exospore. Its protonema is massive with filamentous chloronema formed inside. The terminal part of the chloronema differentiated into filamentous caulonema and its rhizoid was derived from the apical cell of the filamentous chloronema. The initial cell of gametophyte differentiated from chloronema and caulonema. Sporeling-type of Hypnum pacleseens is developmentally similar to Glyphmitrium-type.     

罗汉果大孢子发生及雌配子体发育与花部形态、胚珠变化的关系

为弄清罗汉果(Siraitia grosvenorii)大孢子发生、雌配子体发育过程与花部形态特征、胚珠的关系,运用石蜡切片法对罗汉果子房进行了显微观察。结果表明,罗汉果的胚珠倒生,双珠被,厚珠心,大孢子四分体呈线型排列,合点端一个大孢子分化为功能大孢子,成熟胚囊为蓼型。花蕾形态、胚珠变化与大孢子发生、雌配子体的发育时期具有一定相关性,当子房长度为7.0 mm≤L<9.0 mm,珠心呈椭圆形时,约有45.83%的大孢子母细胞处于减数分裂时期。因此,依据罗汉果花部形态可有效确定大孢子发生与雌配子体发育的时期。     

罗汉松雄球花及其雄配子体发育的形态结构

该研究采用光学显微镜和扫描电镜,观察罗汉松雄球花、小孢子及其配子体发育过程的形态结构特征,以揭示罗汉松小孢子的发生和雄配子体的发育规律,为罗汉松的生殖和杂交组合提供胚胎学证据。结果发现：(1)罗汉松花芽于每年的7月开始分化,至次年5月花粉成熟散粉,雄球花由单生的卵圆形转为2~3个葇荑花序并生,小孢子叶螺旋状着生于圆柱状的花序轴上,每一小孢子叶远轴面基部并列着生2个小孢子囊。(2)小孢子囊壁发育过程中由外及里出现各由1层薄壁细胞组成的表皮、药室内壁、中层和绒毡层,至散粉前,后两者基本被分解吸收。(3)同一小孢子囊内的造孢细胞发育在时间上存在差异,小孢子母细胞减数分裂后形成的四分体有四面体型和十字交叉型两种排列方式,成熟的雄配子体包括生殖细胞和粉管细胞,发育过程中出现的第一和第二原叶细胞大部分被分解消失。(4)电镜下罗汉松花粉粒为典型的松花型花粉,两侧各具1个气囊,远极面具一萌发沟,花粉粒表面具纹理或皱褶。     

Establishment and growth characteristics of a cell suspension culture of Marchantia polymorpha L. with high chlorophyll content

A green-pigmented cell suspension culture of Marchantia polymorpha was established using the medium of Murashige and Skoog with addition of organic acids of the tricarboxylic-acid cycle, vitamins and sugars plus sugar alcohols, exclusion of kinetin, and replacement of sucrose with glucose. In continuous light, the cells grew exponentially for ca. 10 days; in the dark, they grew only to a slight extent. The light-grown cells contained well-developed chloroplasts, and chlorophyll content reached almost twice that of the intact gametophyte.     

Female reproductive development and pollen tube growth in diploid genotypes of<Emphasis Type="Italic"> Solanum cardiophyllum</Emphasis> Lindl.

We have characterized female gametophyte (megagametophyte) development and the kinetics of pollen tube growth in self-pollinated diploid genotypes (2n=2x=24) of Solanum cardiophyllum Lindl. that show normal seed formation. In this species megasporogenesis and megagametogenesis give rise to a female gametophyte of the Polygonum type composed of two synergids, an egg cell, a binucleated central cell and three antipodals; however, asynchronous abnormalities resembling mechanisms that prevail during the formation of second division restitution gametes were observed. In self-pollinated pistils at least 1–2% of germinating pollen tubes were able to reach the megagametophyte 60–84 hours after pollination (hap). Although the egg cell acquired a zygote-like morphology 60–84 hap, division of the primary endosperm nucleus was only observed 84 hap. The analysis of genetic variability in full-sib progeny confirmed that seeds are derived from sexual reproduction. These observations suggest that diploid genotypes of S. cardiophyllum can serve as an ideal system to genetically investigate true seed formation in a tuber-bearing Solanum species.