Effects of Temperature on Induction of Regenerative Stamens in vitro Culture,Microsporogenesis and Pollen Development in Hyacinthus orientalis L.

In order to culture the regenerated stamens of hyacinth in vitro to maturity, the effects of temperature on regeneration of stamens, microsporogenesis and pollen development were studied. Results showed: the proper temperature for stamen regeneration was 25℃. The temperature going down gradually was advantageous to the microsporogenesis and pollen development. The most suitable temperatures for differentiation of microsporocyte, meiotic division and pollen first mitosis were 20–25℃, 20℃ and 10℃ respectively. Under such temperature condition, it was possible to culture the regenerated stamens to maturity, and pollen grains in the stamens had the higher germination frequency. On the contrary, unsuitable temperature condition will make microsporogenesis and pollen development stop at certain development stage, it will finally result in pollen abortion.     

Cytological Observations of Microsporogenesis and Pollen Development in Wheat in Vivo

Cytological observations of microsporogenesis and pollen development in vivo in wheat were carried on by means of phase contrast optics, which could avoid the distortions resulted from materials difficult to be fixed. The cytological changes were observed as follows: 1. Just before first mitosis of pollen, many strands of cytoplasm arose from one side of the nucleus facing the aperture, and moved swingingly toward the aperture. And then these strands of cytoplasm combined into one mass and protruded in the large vacuole. 2. There was a alteration in the direction of the spindle axis from obliquity to parallelism in anaphase. 3. Forming course of the wall between generative and vegetative cell. 4. Dynamic course of the disintegration of wall between vegetative and generative cell as well as that the generative cell came into the vetetafive cell.     

The male germ unit in the pollen and pollen tubes ofPetunia hybrida: Ultrastructural,quantitative and three-dimensional features

Summary The male germ unit ofPetunia hybrida was examined quantitatively and qualitatively at the ultrastructutral level. Three-dimensional reconstructions, the determination of nuclear and cytoplasmic volumes and surface areas, and organelle counts were obtained from serial ultrathin sections and computer analysis. In the mature pollen grain, an elongated generative cell is found in direct physical association with and partially surrounded by the vegetative nucleus. The mature generative cell lacks plastids and has mitochondria equally distributed at both of its tapering ends. In the pollen tube, the sperm cells are physically associated by cytoplasmic connections to each other and to the surrounding vegetative cell membrane. At full style length, the lobed vegetative nucleus and sperm pair are found in close association near the end of the pollen tube. The two sperms of a pair are not strongly dimorphic.     

Ultrastructural features of Aloe ciliaris pollen

Summary Both the internal anatomy and the external morphology of the mature pollen grain of Aloe ciliaris have been studied, together with the cytological changes occurring during pollen activation. In mature pollen, the generative cell (GC) and the vegetative nucleus (VN) are closely associated with each other, and both can be found in the central part of the grain. In the generative cytoplasm, some organelles and microtubular bundles are present. In the vegetative cell, dictyosomes, stacks of rough endoplasmic reticulum, mitochondria, plastids, vacuoles, ribosomes, and masses of fibrillar material have been described. During pollen activation, important changes occur in both the generative and vegetative cells (VC). In the GC, the microtubular bundles become clearly visible, and the GC and VC gradually move towards the germ pore. The RER cisterns become free from the stacks, and organelles, such as dictyosomes, become very active. The fibrillar masses gradually decrease in number, and the individual fibrils become more evident and clearer in resolution.This research was carried out in the framework of contract no. BAP-0204-I of the Biotechnology Action Programme of the Commission of the European Communities     

Ultrastructure of Male Gametophyte in wheat I. The Formation of Generative and Vegetative Cells

The present study of the formation of the generative and vegetative cells in wheat has demonstrated some cytological details at the ultrastructural level. The phragmoplast formed in telophase of the first microsporic mitosis extended centrifugally until it connected with the intine of the pollen grain. A new cell wall was then formed to separate the generative and the vegetative cells. By unequal cytokinesis the former is small and the latter large. In early developmental stage of male gametophyte, the organelles in the cytoplasm of the generaVive cell and the vegetative cells are similar, including mitochondria, dictyosomes, rough endoplasmic retieulum, free and clustered ribosomes and plastids, but microtubules were observed only in the early cytokinesis stage. In the further developmental stage of the male gemetophyte, the generative cell gradually detached from the intine of pollen grain and grew inward to the cytoplasm of the vegetation cell. When the generative cell became round and free in the cytoplasm of the vegetative cell, the wall materials between plasma membranes of the cytoplasm of the generative and the vegetative cells disappeared completely, so that it was a naked cell with a double-layer membrane at this time. The heterogeneity between both cells was then very conspiceous. The organelles in the cytoplasm of the generative cell have hardly any changed besides the degeneration of plastids, but in vegetative cytoplasm the mitochondria and plastids increased dramatically both in number and size. The rapid deposition of starch in the plastids of the cytoplasm of the vegetative cell made the most conspicuous feature of the vegetative cell in mature pollen grain. The significance of the presence of a temporary cell wall in generative cell and heterogeneity between generative and vegetative cells are discussed.     

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.     

土麦冬离体萌发花粉管中生殖细胞与营养核的动态变化

主要报道了土麦冬人工培养萌发花粉管中生殖细胞与营养核的动态变化。多数花粉管中,生殖细胞与营养核贴合后,开始进行有丝分裂,贴合时,营养核略呈弥散状态。在分裂早中期,生殖细胞与营养核分开,从贴合到分开大约经历３－５ｈ,精子形成后,不与营养核连接。ＤＡＰＩ对生殖细胞的有丝分裂有抑制作用。少数花粉管中,生殖细胞核进行无丝分裂,有缢裂和劈裂两种方式。生殖细胞核发生缢裂的花粉管中,未观察到生殖细胞与营养核的贴     

Pollen Bud Formation and its Role in Ophiorrhiza spp.

The anther in Ophiorrhiza is dithecous and tetralocular, itsdevelopment being of the dicotyledonous type. The anther wallcomprises epidermis, endothecium, middle layer and secretorytapetum. The pollen grains are tricolpate and triporate. Themicrospore nucleus undergoes division to form a vegetative nucleusand a generative nucleus and protrusions (pollen buds) are formedfrom the germ pores after the first division of the microsporenucleus. The vegetative nucleus moves into one of these budsor first breaks into a number of irregularly sized nuclear particleswhich enter into one, two or into all three buds, where theydegenerate. Then the pollen buds are separated and the generativenucleus divides inside the pollen tube to form two sperms. Ophiorrhiza harrisiana, Ophiorrhiza hirsutula, microsporogenesis, pollen buds, vegetative nucleus     

Detection and quantification of rRNA by high-resolutionin situ hybridization in pollen grains

We examined changes in the localization of cytoplasmic rRNA during pollen development inNicotiana tabacum SR-1. The rRNA was visualized byin situ hybridization, and the signal intensity of rRNA in microspore, vegetative and generative cell was quantified by microphotometry. The amount of rRNA per microspore or pollen section increased about 5 times from microspore to mature pollen grain and kept increasing even in the late stage of pollen development after PMI. The increase of rRNA occur in both vegetative and generative cells. The results suggest that synthesis of rRNA occur even after PM I in both vegetative and generative cells.     

杜仲花粉不对称分裂的超微结构观察

运用透射电镜对杜仲花粉发育进程进行了观察研究。结果显示,杜仲小孢子的第一次分裂为不等分裂,形成小的生殖细胞和大的营养细胞。分裂开始前小孢子的营养极形成许多小液泡,建立细胞极性;然后随着核膜的解体核周围的细胞器逐渐向纺锤体区靠近,围绕在纺锤体周围。花粉第一次有丝分裂完成后,生殖细胞所获得的细胞器开始分布在细胞的两侧,后来移向生殖细胞的营养极,而紧贴花粉壁的生殖极无细胞器分布。这种生殖细胞早期的细胞极性,可能为进一步分裂形成精细胞奠定基础。     

青葙花药发育的结构和组织化学观察

对苋科植物青葙Celosia argentea花药发育的结构和组织化学（多糖和脂滴）特征进行观察。青葙小孢子发生为同时型,四分体为四面体型。药壁为典型四层,绒毡层属于同型绒毡层。成熟花粉为二胞型。早期花药中的淀粉粒和脂滴均较少,绒毡层细胞至小孢子晚期退化为体积较大的脂块。二胞花粉时期的中层细胞退化为脂滴。早期二胞花粉中先出现多糖颗粒,晚期的成熟花粉中积累大量淀粉粒和较少的脂滴为营养储存物。     

Monitoring by epifluorescence microscopy of organelle DNA fate during pollen development in five angiosperm species

The fates of mitochondrial and plastid nucleoids during pollen development in six angiosperm species (Antirrhinum majus, Glycine max, Medicago sativa, Nicotiana tabacum, Pisum sativum, and Trifolium pratense) were examined using epifluorescence microscopy after double staining with 4',6-diamidino-2- phenylindole (DAPI) to stain DNA and with a potentiometric dye (either DiOC7 or rhodamine 123) for visualization of metabolically active mitochondria. From the pollen mother cell stage to the microspore stage of pollen development, mitochondria and plastids both contained DNA detectable by DAPI staining. However, during the further maturation preceding anthesis, mitochondrial DNA became undetectable cytologically in either the generative or the vegetative cell of mature pollen; even in germinated pollen tubes containing hundreds of metabolically active mitochondria undergoing cytoplasmic streaming, vital staining with DAPI failed to reveal mitochondrial DNA. By the mature pollen stage, plastid DNA also became undetectable by DAPI staining in the vegetative cell. However, in the generative cell of mature pollen the timing of plastid DNA disappearance as detected by DAPI varied with the species. Plastid DNA remained detectable only in the generative cells of pollen grains from species known or suspected to have biparental transmission of plastids. The apparent absence of cytologically detectable organelle genomes in living pollen was further examined using molecular methods by hybridizing organelle DNA-specific probes to digests of total DNA from mature pollen and from other organs of A. majus and N. tabacum, both known to be maternal for organelle inheritance. Mitochondrial DNA was detected in pollen of both species; thus the cytological alteration of mitochondrial genomes during pollen development does not correspond with total mtDNA loss from the pollen. Plastid DNA was detectable with molecular probes in N. tabacum pollen but not in A. majus pollen. Since the organelle DNA detected by molecular methods in mature pollen may lie solely in the vegetative cell, further study of the basis of maternal inheritance of mitochondria and plastids will require molecular methods which distinguish vegetative cell from reproductive cell organelle genomes. The biological effect of the striking morphological alteration of organelle genomes during later stages of pollen development, which leaves them detectable by molecular methods but not by DAPI staining, is as yet unknown.     

楸树大小孢子发生与雌雄配子体发育的研究

运用石蜡切片法和整体透明法对楸树(Catalpa bungei C.A.Meyer)大、小孢子发生及雌、雄配子体发育过程进行了研究.结果表明:楸树可育雄蕊2枚,花药4室,药壁发育属双子叶型,腺质异型绒毡层.小孢子母细胞减数分裂为同时型,四分体后小孢子不分离形成正四面体型四合花粉,偶有左右对称型和十字交叉型.成熟花粉为二细胞型,无萌发孔.子房上位,2室,中轴胎座,胚珠多数,倒生,单珠被,薄珠心,具珠被绒毡层.单孢原直接发育为大孢子母细胞,四分体线形排列,合点端大孢子发育为功能大孢子,胚囊发育为蓼型.雄蕊发育早于雌蕊,花开后雌、雄蕊趋于同熟.研究认为:虽然楸树雌、雄蕊发育过程中均存在一定比例的败育,但其花而不实"并非雄性或雌性不育所致.推测与其授粉受精和胚后发育有关.     

Cytological and Cytochemical Studies of Temperature Induced Male Sterility in Taihangia rupestris

Using in vitro-flower experimental system of Taihangia rupestris Yü et Li in which development or abortion of stamens could be controlled by temperature, the cytological and cytochemical changes in both development and abortion processes of the stamens were comparatively studied. Cytological changes of the stamen abortion initiated at the microsporocyte stage before which normal cytological process were seen from stamen primordium to primary microsporocyte stage. However, despite of the normal cytological process, polysaccharide accumulation was never observed in the cells of anther and filament where as in the normally developing stamens polysaccharide accumulation must largely occurred in those cells mentioned above. The author considered that such cytochemical changes prior to the morphological deviances might be of great significance to approach the cause of tempera-ture-induced male sterility.     

Epigenetic marks in the <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L. mature pollen grain and during in vitro pollen tube growth

During the sexual reproduction of flowering plants, epigenetic control of gene expression and genome integrity by DNA methylation and histone modifications plays an important role in male gametogenesis. In this study, we compared the chromatin modification patterns of the generative, sperm cells and vegetative nuclei during Hyacinthus orientalis male gametophyte development. Changes in the spatial and temporal distribution of 5-methylcytosine, acetylated histone H4 and histone deacetylase indicated potential differences in the specific epigenetic state of all analysed cells, in both the mature cellular pollen grains and the in vitro growing pollen tubes. Interestingly, we observed unique localization of chromatin modifications in the area of the generative and the vegetative nuclei located near each other in the male germ unit, indicating the precise mechanisms of gene expression regulation in this region. We discuss the differences in the patterns of the epigenetic marks along with our previous reports of nuclear metabolism and changes in chromatin organization and activity in hyacinth male gametophyte cells. We also propose that this epigenetic status of the analysed nuclei is related to the different acquired fates and biological functions of these cells.     

Identificaton and characterization of stamen- and tapetum-specific genes from tomato

Summary Differential screening of a tomato cDNA library produced from pre-anthesis stamens resulted in the isolation of 25 cDNA clones that hybridized to probes made from stamen RNA and showed no hybridization to probes made from RNA of vegetative organs. The 25 clones were found to represent 11 noncross-hybridizing classes. The majority of these clones were derived from genes that were single or low copy in the tomato genome. Northern RNA blotting experiments of vegetative and floral organs at several stages of development demonstrated that expression in all 11 classes was confined to floral organs. Of the 11 classes 9 were found to be expressed exclusively in stamens prior to anthesis. Two classes showed expression in immature stamens and in petals, with one of these two additionally being expressed in mature stamens at anthesis. Clones from three of the classes that were expressed exclusively in stamens were used as probes for in situ localization of RNA in floral organs. These experiments demonstrated that expression of the genes corresponding to these clones was confined to the tapetal cells of the anthers. Expression of one of the three genes was found to be limited to a single cell type during the 5–6 day period from late meiosis to immature pollen formation.     

Fluorescence microscopy of plastid nucleoids and a survey of nuclease C in higher plants with respect to mode of plastid inheritance

Summary Plastid nucleoids (pt nucleoids) were observed during pollen formation, or in generative cells of mature pollen grains using fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI). Nuclease C activity was surveyed using SDS-PAGE and agarose gel nuclease assay methods. InMirabilis jalapa, pt nucleoids were observed both in pollen mother cells and the monocellular pollen grains after meiosis, followed by the complete disappearance both in the generative and vegetative cells at the bicellular pollen grain stage. This observation is a direct evidence of maternal plastid inheritance. By contrast, in the generative cells of mature pollen grains fromRhododendron kaempferi, Zygocactus truncatus, Oenothera laciniata, andO. speciosa, pt nucleoids were clearly observed. Thus cytological evidence convinces the mode of biparental plastid inheritance. Nuclease C activity was clearly detected both in the stamen and pistil ofM. jalapa. InR. kaempferi low nuclease C activity was detected in both organs, but the activity in the stamen was much less than in the pistil. InZ. truncatus, O. laciniata, andO. speciosa, the activities were difficult to detect in both organs. These results suggest a significant role of nuclease C for the digestion of pt nucleoids in the generative cells.Abbreviations EGTA ethylene-glycol-bis-(2-aminoethyl ether)-N, N, N, N-tetraacetic acid - DAPI 4,6-diamidino-2-phenylindole - Nuclease C Ca²⁺ dependent nuclease - SDS-PAGE SDS-polyacrylamide gel electrophoresis - pt nucleoids plastid nucleoids     

Anther culture of Sorghum bicolor (L.) Moench

The sequence of pollen development from the tetrad stage to the mature tricellular grain was studied in freshly harvested anthers of Sorghum bicolor. This pattern of development was then compared with that occurring during panicle pretreatment and subsequent anther incubation in vitro. It was found that during pretreatment at 7° C mitoses of the vegetative cell were induced in up to 30% of the pollen. During anther incubation procallus development was highly polarised with contributions from both the generative and vegetative cells. After pretreatment at 14 or 20° C the generative cell became detached from the pollen wall and it was not possible to determine whether subsequent development involved only the vegetative cell or both the vegetative and generative cells.Although retarded pollen grains were observed both in vivo and in vitro, and were occasionally seen to divide in culture, they did not appear to be the source of the procalluses produced.     

Juxtaposition of the generative cell and vegetative nucleus in the mature pollen grain of amaryllis (Hippeastrum vitatum)

Summary Computer-generated, three-dimensional reconstructions from serial ultrathin sections were used to investigate the spatial organization and extent of association between the generative cell and vegetative nucleus within the mature pollen grain of amaryllis. In all cases examined, the highly lobed vegetative nucleus was found in close proximity and positioned laterally to the elongated, oval shaped generative cell. Numerous projections of the vegetative nucleus come to within 53 nm of the inner vegetative cell plasma membrane which surrounds the generative cell. These areas of close association may continue transversely around the generative cell for a distance of up to 4 m. Although an association exists between the generative cell and vegetative nucleus of the mature pollen grain, it is apparent that several changes must take place after pollination in order to achieve the high amount of close contact that occurs between the vegetative nucleus and the numerous terminal cell extensions of the leading sperm in the pollen tube of amaryllis (Mogensen 1986). Thus, this study demonstrates that the spatial organization among components of the male germ unit in the mature pollen grain does not necessarily reflect relationships that ultimately exist among these components within the pollen tube.