Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.     

Microspore development inBrassica napus and the effect of high temperature on division in vivo and in vitro

Summary Brassica napus cv. Topas microspores isolated and cultured near the first pollen mitosis and subjected to a heat treatment develop into haploid embryos at a frequency of about 20%. In order to obtain a greater understanding of the induction process and embryogenesis, transmission electron microscopy was used to study the development of pollen from the mid-uninucleate to the bicellular microspore stage. The effect of 24 h of high temperature (32.5 °C) on microspore development was examined by heat treating microspore cultures or entire plants. Mid-uninucleate microspores contained small vacuoles. Late-uninucleate vacuolate microspores contained a large vacuole. The large vacuole of the vacuolate stage was fragmented into numerous small vacuoles in the late-uninucleate stage. The late-uninucleate stage contained an increased number of ribosomes, a pollen coat covering the exine and a laterally positioned nucleus. Prior to the first pollen mitosis the nucleus of the lateuninucleate microspore appeared to be appressed to the plasma membrane; numerous perinuclear microtubules were observed. Microspores developing into pollen divided asymmetrically to form a large vegetative cell with amyloplasts and a small generative cell without plastids. The cells were separated by a lens-shaped cell wall which later diminished. At the late-bicellular stage the generative cell was observed within the vegetative cell. Starch and lipid reserves were present in the vegetative cell and the rough endoplasmic reticulum and Golgi were abundant. The microspore isolation procedure removed the pollen coat, but did not redistribute or alter the morphology of the organelles. Microspores cultured at 25 °C for 24 h resembled late-bicellular microspores except more starch and a thicker intine were present. A more equal division of microspores occurred during the 24 h heat treatment (32.5 °C) of the entire plant or of cultures. A planar wall separated the cells of the bicellular microspores. Both daughter cells contained plastids and the nuclei were of similar size. Cultured embryogenie microspores contained electron-dense deposits at the plasma membrane/cell wall interface, vesicle-like structures in the cell walls and organelle-free regions in the cytoplasm. The results are related to embryogenesis and a possible mechanism of induction is discussed.Abbreviations B binucleate - LU late uninucleate - LUV late uninucleate vacuolate - M mitotic - MU mid-uninucleate - RER rough endoplasmic reticulum - TEM transmission electron micrograph     

ULTRASTRUCTURE OF ANOMALOUS POLLEN DEVELOPMENT IN EMBRYOGENIC ANTHER CULTURES OF HYOSCYAMUS NIGER

The formation of anomalous, binucleate pollen grains and their subsequent embryogenic development, induced by anther culture in Hyoscyamus niger, were analyzed by transmission electron microscopy (TEM). In culture, uninucleate pollen grains occasionally divided symmetrically giving rise to two apparently identical nuclei sharing a common cytoplasm. These nuclei divided once or twice unaccompanied by cell wall formation. After the daughter nuclei organized into cells, their subsequent division products contributed to embryoid formation. In conjunction with previous studies of pollen embryogenesis in H. niger, it appears that in contrast to the principle mode of embryogenesis (i.e., first asymmetric division forms typical two-celled pollen grain and the generative cell acts as the embryogenic precursor), anomalous pollen show no carry-over of gametophytic influences following embryogenic induction. This suggests that specific pathways of embryogenesis are correlated with the rate at which gametophytic gene activity is repressed following induction.     

Pollen ultrastructure in anther cultures of Datura innoxia. III. Incomplete microspore division.

During the microspore division in Datura innoxia, the mitotic spindle is oriented in planes both perpendicular (PE) and oblique (OB) to the spore wall against which the nucleus is situated. However, irrespective of polarity, the usual type of hemispherical wall is laid down at cytokinesis and isolates the generative cell from the rest of the pollen grain (type A). In PE spores the vegetative nucleus initially occupies a central position in the pollen grain, whereas in OB spores the vegetative nucleus lies at the periphery of the grain close to the generative cell. In anther cultures initiated just before the microspore division is due to take place, no marked change can be observed in either orientation or symmetry of the mitotic spindle when the spores divide. In some, however, cytokinesis is disrupted and deposition of the hemispherical wall arrested. In the absence of a complete wall, differentiation of the generative cell cannot take place and binucleate pollen grains are formed having 2 vegetative-type nuclei (type B). The 2 nuclei in the B pollens are always situated against the pollen-grain wall, suggesting that the disruption phenomenon is related to the OB spores. The incomplete wall always makes contact with the intine on the intine-side of the spindle. Wall material may be represented merely as short stubs projecting out from the intine into the cytoplasm, in which event the 2 nuclei lie close to each other and are separated by only a narrow zone of cytoplasm. In other grains the wall is partially developed between the nuclei and terminates at varying distances from the tonoplast; in these, the nuclei are separated by a wider zone of cytoplasm. The significance of these binucleate grains in pollen embryogenesis is discussed.     

Cytoskeletal changes and induction of embryogenesis in microspore and pollen cultures of Brassica napus L.

Microspores and pollen of Brassica napus were cultured under conditions leading to embryo formation. Concomitant changes in cytoskeletal configurations were analysed. The microfilamental cytoskeleton exhibited a loss of polarity in embryogenic cells but cytochalasin treatment revealed that microfilaments do not influence embryogenesis. Two embryogenic pathways started from microspores and were either characterized by turned division planes or by division when the nucleus was in the cell centre. In both cases microtubules clearly exhibited new arrangements and likely played a major role in newly induced symmetrical division. In pollen, embryogenic development started in the vegetative cell provided the generative cell was arrested near the pollen wall. The concomitant disappearance of defined microtubular arrays is likely to be responsible for the positioning of the cell.     

Differentiation of generative and vegetative cells in angiosperm pollen

 Cellular differentiation of a generative and a vegetative cell is an important event during microspore and pollen development and is requisite for double fertilization in angiosperms. The generative cell produces two sperm cells, or male gametes, whereas the vegetative cell produces an elongated pollen tube, a gametophytic cell, to deliver the male gametes to the embryo sac. For typical differentiation of the gametic and gametophytic cells, cell polarity, including nuclear positioning, must be established prior to microspore mitosis and be maintained during mitosis. Microtubules are closely involved in the process of asymmetric cell division. On the other hand, alteration of the chromatin composition seems to be responsible for the differential gene expression between the generative and vegetative cells. Cytoplasmic regulatory molecules, which affect chromatin configuration, are postulated to be unequally distributed to the two cells at the asymmetric cell division. Thus, typical differentiation of the cells is accomplished by a cellular mechanism and a molecular mechanism, which might be independent of each other. These results are discussed in relation to one model that accounts for the different fates of generative and vegetative cells during sexual plant reproduction. Received: 3 September 1996 / Revision accepted: 23 September 1996     

莴苣花粉发育过程中ATP酶的分布

对莴苣花粉发育过程中ATPase的分布特征做了研究。四分体早期的小孢子细胞质中开始出现ATPase反应颗粒。之后,小孢子在发育过程中,花粉内壁聚集大量体积较大的ATPase反应颗粒,并一直保持到花粉即将成熟。在小孢子发育晚期,在花粉萌发孔处和小孢子大液泡中也特异性地聚集了较多ATPase颗粒。二胞花粉刚形成的生殖细胞表面呈现大量的ATPase反应颗粒,当生殖细胞脱离花粉内壁移入营养细胞,ATPase反应颗粒基本消失。生殖细胞分化过程中生殖细胞的ATPase反应颗粒逐渐低于营养细胞中的。在成熟花粉中,精细胞中的ATPase反应颗粒比营养细胞中的少,且主要集中在细胞核中。结果显示花粉发育过程中ATPase的特异分布与花粉发育的一些生物学事件密切相关。     

A CYTOLOGICAL ANALYSIS OF MICROSPORES OF TRITICUM AESTIVUM (POACEAE) DURING NORMAL ONTOGENY AND INDUCED EMBRYOGENIC DEVELOPMENT

Uninucleate microspores of Triticum aestivum cv. Pavon can be induced in vitro to alter their development to produce embryoids rather than pollen. Microspores expressed their embryogenic capacity through one of two division pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be a typical bicellular pollen grain. Here the generative cell detached from the intine, migrated to a central position in the pollen grain, and underwent a second haploid mitosis as the vegetative cell divided to give rise to the embryoid. In the second pathway, the first division was symmetric and both nuclei divided repeatedly to form the embryoid. This comparative analysis of normal pollen ontogeny and induced embryogenesis provided no evidence for the existence of predetermined embryogenic microspores in vitro or in vivo. Instead, microspores are induced at the time of culture, and embryogenesis involves continued metabolic activity associated with the gradual cessation of the gametophytic pathway and a redifferentiation into the sporophytic pathway. In conjunction with a previous study, it appears that embryogenic induction of wheat microspores involves switching off gametophytic genes and derepressing sporophytic genes.     

Maintenance of gametophytic development after symmetrical division in tobacco microspore culture

Isolated tobacco (Nicotiana tabacum L.) microspores maturing in vitro can be induced to undergo symmetrical divisions, instead of the normal asymmetrical first pollen mitosis, by addition of anther extracts to the culture medium. The two daughter cells in symmetrically divided pollen resemble vegetative pollen cells in cytological characteristics, nuclear size and chromatin condensation, are separated by a cell wall and remain viable during in vitro maturation. After transfer to a germination medium, only one of the two vegetativelike cells forms a pollen tube in vitro. Therefore, apparently normal gametophytic development can be maintained after symmetrical microspore division. These results are discussed in relation to current models for induction of microspore embryogenesis.     

An autoradiographic study of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger

Summary The pattern of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger was studied using³H-uridine autoradiography. Incorporation of label during pollen maturation was periodic with peak RNA synthesis occurring in the uninucleate, nonvacuolate pollen grains and in the vegetative cell of the bicellular pollen grains. During the early stages of germination, isotope incorporation occurred predominantly in the nucleus of the vegetative cell with little or no incorporation in the generative cell. With the appearance of the pollen tube, incorporation of³H-uridine in the vegetative cell nucleus decreased and completely disappeared at later stages of germination. No incorporation of isotope was observed in the sperms formed in the pollen tube by the division of the generative cell. From a comparison of the results of this study with those of previous works on RNA synthesis during pollen embryogenesis in cultured anthers ofH. niger, it is concluded that in contrast to embryogenic development, there is no requirement for sustained RNA synthesis by the generative cell nucleus for normal gametophytic development.     

Nuclear fusion in cultured microspores of barley

Fusion of the generative and vegetative nuclei physically separated by a wall has been observed in cultured microspores of barley. The generative cell appears to play an active role in fusion as it elongates toward the vegetative nucleus, becomes detached from the microspore wall, and finally completely encloses the vegetative nucleus. The generative cell wall disappears before nuclear fusion takes place. Since these events have been known to occur during pollen development in vivo, it is hypothesized that the occurrence of nuclear fusion in cultured microspores is the result of continued expression of the genes for gametophytic development.     

含笑花药发育的超微结构研究

对含笑花药发育中的超微结构变化进行观察,结果显示:(1)花粉发育中有三次液泡变化过程——第一次是小孢子母细胞在形成时内部出现了液泡,这可能与胼胝质壁的形成有关;第二次是在小孢子母细胞减数分裂之前,细胞内壁纤维素降解区域形成液泡,它的功能可能是消化原有的纤维素细胞壁;第三次是在小孢子液泡化时期,形成的大液泡将细胞核挤到边缘,产生极性。(2)含笑花粉在小孢子早期形成花粉外壁外层,花粉外壁内层在小孢子晚期形成,而花粉内壁是在二胞花粉早期形成;花粉成熟时,表面上沉积了绒毡层细胞的降解物而形成了花粉覆盖物。研究认为,含笑花粉原外壁的形成可能与母细胞胼胝质壁有关,而由绒毡层细胞提供的孢粉素物质按一定结构建成了花粉覆盖物。     

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。     

Cellular changes during the acquisition of embryogenic potential in isolated pollen grains ofNicotiana tabacum

Summary We used electron microscopical techniques to study ultrastructural changes during the acquisition of embryogenic competence in immature pollen grains ofNicotiana tabacum, isolated at the early- or mid-bicellular stage and cultured in vitro under starvation conditions. Cytoplasmic and nuclear changes during the starvation treatment are reported. Dedifferentiation of plastids, dilation of the wall of the generative cell, the appearance of a large vacuole, loss of nuclear pores in the vegetative nucleus, changes in chromatin and nucleolar structure, and a decrease in the size of the nucleolus were observed. We suggest that these events are the first step in the switch from generative to vegetative generation during pollen embryogenesis.     

Ultrastructural studies on plastids of generative and vegetative cells inLiliaceae 3. Plastid distribution during the pollen development inGasteria verrucosa (Mill.) Duval

Summary This paper describes the development of pollen grains ofGasteria verrucosa from the late microspore to the mature two-cellular pollen grain. Ultrastructural changes and the distribution of plastids as a result of the first pollen mitosis have been investigated using light and electron microscopy. The microspores as well as the generative and the vegetative cell contain mitochondria and other cytoplasmic organelles during all of the observed developmental stages. In contrast, the generative cell and the vegetative cell show a different plastid content. Plastids are randomly distributed within the microspores before pollen mitosis. During the prophase of the first pollen mitosis the plastids become clustered at the proximal pole of the microspore. The dividing nucleus of the microspore is located at the distal pole of the microspore. Therefore, the plastids are not equally distributed into both the generative and the vegetative cell. The possible reasons for the polarization of plastids within the microspore are briefly discussed. The lack of plastids in the generative cell causes a maternal inheritance of plastids inGasteria verrucosa.     

Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis

Specific stress treatments (sucrose starvation, alone or combined with a heat shock) applied to isolated tobacco (Nicotiana tabacum L.) microspores irreversibly blocked normal gametophytic development and induced the formation of embryogenic cells, which developed subsequently into pollen-derived embryos by culture at 25°C in a sugar-containing medium. A cold shock at 4°C did not inhibit microspore maturation in vitro and did not induce cell division activity, even when combined with a starvation treatment. In the absence of sucrose, microspores isolated in the G1 phase of the cell cycle replicated their DNA and accumulated in G2. Late microspores underwent miotosis during the first day of culture which resulted in a mixed population of bicellular pollen grains and uninucleate microspores, both embryogenic. After the inductive stress treatments the origin of the first multicellular structures, formed in the sugar-containing medium, could be traced to divisions of the microspore cell or divisions of the vegetative cell of bicellular pollen, indicating that the symmetry of microspore mitosis in vitro is not important for embryogenic induction. These results represent a step forward towards a unified model of induction of embryogenesis from microspores/pollen which, within a relatively wide developmental window, are competent to deviate from normal gametophytic development and initiate the alternative sporophytic programme, in response to specific stress signals.Abbreviation DAPI 4,6-diamidino-2-phenylindole We acknowledge the help of Monica Boscaiu and Zarko Hrzenjak with the artwork, and Michaela Braun-Mayer for growing the tobacco plants. This project was financed by the Austrian Fonds zur Forderung der wissenschaftlichen Forschung, grant S6003-BIO.     

Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis

Summary Microspectrophotometry following Feulgen staining and autoradiography following (³H)-thymidine labelling were used to study cell-cycle events during pollen development in tobacco (Nicotiana tabacum L.). During normal gametophytic pollen development in the anther and in vitro the generative nucleus passes through the S phase to the G₂ phase soon after microspore mitosis, while the vegetative nucleus remains arrested in G₁ (=G₀). During embryogenie induction by an in vitro starvation treatment of immature pollen ongoing DNA replication in the generative nucleus is completed and followed by DNA replication in the vegetative cell in a large fraction of the pollen grains. Addition of the DNA replication inhibitor hydroxyurea to the starvation medium postpones S phase entry until the pollen is transferred to a rich medium and does not affect embryo formation. These results demonstrate that one of the crucial events of embryogenic induction is the derepression of the G₁ arrest in the cell cycle of the vegetative cell.     

Generative cell division and sperm cell formation in barley

Summary Shortly before and during division, the generative cell of barley (Hordeum vulgare L.) is located near the vegetative nucleus, in the peripheral layer of the highly vacuolated vegetative cell at the aperture pole. This position is also characteristic of the two resulting sperm cells. Conventional mitosis of the generative cell is followed by cytokinesis through cell plate formation. Just after division, the two sperm cells are enclosed together within a common inner vegetative cell plasma membrane, and they gradually separate from each other only during pollen maturation. The space between the generative or sperm cell plasma membrane and the vegetative cell plasma membrane is very thin and appears to be devoid of a cell wall. Both the generative cell and the young sperm cells contain a normal set of organelles; plastids devoid of starch are only sporadically observed. Our data indicate that in Hordeum vulgare the generative cell divides after migrating inside the pollen grain. This follows the pattern of development well established for several species with tricellular pollen.     

DEVELOPMENT OF OMNIAPERTURATE POLLEN IN TRILLIUM KAMTSCHATICUM (LILIACEAE)

Ultrastructural changes during omniaperturate pollen development in Trillium kamtschaticum Pall, was examined using transmission electron microscopy. The pollen mother cells are not enveloped within a thick callosic wall. The microspores resulting from successive meiosis are divided by scanty deposition of callosic wall in the tetrad. A primexine/exine template is not recognizable within the tetrad during formation of exinous components. Preexinous globules, originating from vesicles in the callosic wall, accumulate electron-dense materials and develop into exinous globules. The preexinous globules have ca 10 nm wide contacts with tilted and invaginated plasma membrane of the microspore within the callosic wall. After dissolution of the callosic wall, the microspores separate and mitosis subsequently leads to the formation of a generative cell and vegetative cell encased in a loose aggregation of developing exinous globules. When the generative cell is at the pollen grain surface, the channeled zone is initiated at the opposite side of the microspore on the surface of the vegetative cell. Just before pollen maturity, a new layer develops under the channeled zone. Thus, development of the omniaperturate pollen grains of T. kamtschaticum involves some processes that are distinct from those of Canna and Heliconia and some that are similar.