Structural changes during the first divisions of embryos resulting from anther and free microspore culture inBrassica napus (original) (raw)
Related papers
Annals of Botany, 1998
A comparative morphological study of microspore-derived (MD) and zygotic embryos of Brassica napus L. was conducted, illustrating substantial similarities in external morphology of these embryos throughout their development. Haploid embryos were produced from isolated microspores cultured on high molecular weight polyethylene glycol (PEG), replacing sucrose as an osmoticum. Morphological changes during the time-course of microspore embryo development induced on PEG (25 %) and sucrose (13 %) are described in detail as revealed by scanning electron microscopy (SEM) and compared to the corresponding stages of zygotic embryos developed in o ulo. At the heart, torpedo and early cotyledonary stages, microspore-derived (MD) embryos on PEG closely resemble their zygotic counterparts. In contrast, the external morphology of embryos induced on high sucrose medium differs from that of PEG and zygotic embryos indicating that a high concentration of sucrose in culture has a morphogenetic effect on MD embryo development in B. napus. Fragments of the original pollen wall are regularly observed at the root pole region and at the tips of suspensors in MD embryos throughout their development. This suggests that polarity in MD embryos might originate from structurally polarized late uninuclear microspores and early bicellular pollen.
Protoplasma, 1995
In the present study, microsporogenesis, microgametogenesis and pollen wall ontogeny in Campsis radicans (L.) Seem. were studied from sporogenous cell stage to mature pollen using transmission electron microscopy. To observe the ultrastructural changes that occur in sporogenous cells, microspores and pollen through progressive developmental stages, anthers at different stages of development were fixed and embedded in Araldite. Microspore and pollen development in C. radicans follows the basic scheme in angiosperms. Microsporocytes secrete callose wall before meiotic division. Meiocytes undergo meiosis and simultaneous cytokinesis which result in the formation of tetrads mostly with a tetrahedral arrangement. After the development of free and vacuolated microspores, respectively, first mitotic division occurs and two-celled pollen grain is produced. Pollen grains are shed from the anther at two-celled stage. Pollen wall formation in C. radicans starts at tetrad stage by the formation of exine template called primexine. By the accumulation of electron dense material, produced by microspore, in the special places of the primexine, first of all protectum then columellae of exine elements are formed on the reticulatepatterned plasma membrane. After free microspore stage, exine development is completed by the addition of sporopollenin from tapetum. Formation of intine layer of pollen wall starts at the late vacuolated stage of pollen development and continue through the bicellular pollen stage.
Identification of potentially embryogenic microspores in Brassica napus
Physiologia Plantarum, 1988
Studies were undertaken with Brassica napus L. cv. Topas to identify buds containing microspores predisposed to embryogenesis in vitro and to investigate bud and microspore development in relation to this process. No significant correlation was found between the final embryo number and bud components. There appears to be a developmental window of less than 8 h duration during which microspores are very likely to form embryos: over 70% of the microspores can undergo division and up to 70% of these can form embryos. Embryos were mainly obtained from late uninuucleate to early binucleate microspores: the former contained mainly a G2 or M phase nucleus located at the microspore periphery and the latter a generative nucleus (associated with the intine) and a vegetative nucleus. Observations indicated that only the vegetative nucleus contributed to embryo formation. The first embryogenic division occurred between 8 and 16 h for uninucleate-and between 8 and 48 h for binucleate-derived embryos.
Protoplasma, 2014
Isolated microspores of B. napus in culture change their developmental pathway from gametophytic to sporophytic and form embryo-like structures (ELS) upon prolonged heat shock treatment (5 days at 32°C). ELS express polarity during the initial days of endosporic development. In this study, we focussed on the analysis of polarity development of ELS without suspensor. Fluorescence microscopy and 3-D confocal laser scanning microscopy (CLSM) without tissue interfering enabled us to get a good insight in the distribution of nuclei, mitochondria and endoplasmic reticulum (ER), the architecture of microtubular (MT) cytoskeleton and the places of 5-bromo-2′-deoxy-uridine (BrdU) incorporation in successive stages of microspore embryogenesis. Scanning electron microscopy (SEM) analysis revealed, for the first time, the appearance of a fibrillar extracellular matrix-like structure (ECM-like structure) in androgenic embryos without suspensor. Two types of endosporic development were distinguished based upon the initial location of the microspore nucleus. The polarity of dividing and growing cells was recognized by the differential distributions of organelles, by the organization of the MT cytoskeleton and by the visualization of DNA synthesis in the cell cycle. The directional location of nuclei, ER, mitochondria and starch grains in relation to the MTs configurations were early polarity indicators. Both exine rupture and ECMlike structure on the outer surfaces of ELS are supposed to stabilize ELS's morphological polarity. As the role of cell polarity during early endosporic microspore embryogenesis in apical-basal cell fate determination remains unclear, microspore culture system provides a powerful in vitro tool for studying the developmental processes that take place during the earliest stages of plant embryogenesis.
Frontiers in Plant Science, 2015
The induction of microspore embryogenesis produces dramatic changes in different aspects of the cell physiology and structure. Changes at the cell wall level are among the most intriguing and poorly understood. In this work, we used high pressure freezing and freeze substitution, immunolocalization, confocal, and electron microscopy to analyze the structure and composition of the first cell walls formed during conventional Brassica napus microspore embryogenesis, and in cultures treated to alter the intracellular Ca 2+ levels. Our results revealed that one of the first signs of embryogenic commitment is the formation of a callose-rich, cellulose-deficient layer beneath the intine (the subintinal layer), and of irregular, incomplete cell walls. In these events, Ca 2+ may have a role. We propose that abnormal cell walls are due to a massive callose synthesis and deposition of excreted cytoplasmic material, and the parallel inhibition of cellulose synthesis. These features were absent in pollen-like structures and in microspore-derived embryos, few days after the end of the heat shock, where abnormal cell walls were no longer produced. Together, our results provide an explanation to a series of relevant aspects of microspore embryogenesis including the role of Ca 2+ and the occurrence of abnormal cell walls. In addition, our discovery may be the explanation to why nuclear fusions take place during microspore embryogenesis.
Protoplasma, 2012
Isolated microspores and pollen suspension of Brassica napus "Topas" cultured in NLN-13 medium at 18°C follow gametophytic pathway and develop into pollen grains closely resembling pollen formed in planta. This culture system complemented with whole-mount immunocytochemical technology and novel confocal laser scanning optical technique enables detailed studies of male gametophyte including asymmetric division, cytoskeleton, and nuclear movements. Microtubular cytoskeleton configurationally changed in successive stages of pollen development. The most prominent role of microtubules (MTs) was observed just before and during nuclear migration at the early and mid-bi-cellular stage. At the early bi-cellular stage, parallel arrangement of cortical and endoplasmic MTs to the long axis of the generative cell (GC) as well as MTs within GC under the plasmalemma bordering vegetative cell (VC) were responsible for GC lens shape. At the beginning of the GC migration, endoplasmic microtubules (EMTs) of the VC radiated from the nuclear envelope. Most cortical and EMTs of the VC were found near the sporoderm. At the same time, pattern of MTs observed in GC was considerably different. Multiple EMTs of the GC, previously parallel aligned, reorganized, and start to surround GC, forming a basket-like structure. These results suggest that EMTs of GC provoke changes in GC shape, its detachment from the sporoderm, and play an important role in GC migration to the vegetative nucleus (VN). During the process of migration of the GC to the VC, multiple and thick bundles of MTs, radiating from the cytoplasm near GC plasma membrane, arranged perpendicular to the narrow end of the GC and organized into a "comet-tail" form. These GC "tail" MTs became shortened and the generative nucleus (GN) took a ball shape. The dynamic changes of MTs accompanied polarized distribution pattern of mitochondria and endoplasmic reticulum. In order to confirm the role of MTs in pollen development, a "wholemount" immunodetection technique and confocal laserscanning microscopy was essential.
Developmental studies of cytoplasmic male-sterile Brassica napus lines
2005
The presence of male sterile plants in the wild gained the attention of breeders to utilize the trait for hybrid seed production in economically important crops. For scientists, cytoplasmic male sterile (CMS) plants are regarded as an excellent tool to study the genetic interactions between mitochondria and nucleus in flower development. CMS plants can be obtained after sexual crosses between different species of the same family or by somatic hybridisations between unrelated species. The Brassica napus CMS lines investigated in this thesis were obtained after protoplast fusion between B. napus cv. Hanna and Arabidopsis thaliana var. Landsberg erecta. After several backcrosses using B. napus as the male parent, the cells of the CMS lines contain the nucleus and the plastids from B. napus while the mitochondria have a rearranged mitochondrial (mt) DNA between the two species. The vegetative and flower development of the two CMS lines was compared with B. napus. The CMS plants showed a reduced seedling growth. This slower growth rate was present throughout the vegetative development of the CMS plants. They also bolted later than B. napus. However, when fully matured they were of the same size as B. napus. The reduced number of stem cells and its smaller size during the first six weeks of growth seems to be the reason for the shorter stature of the CMS plants. Metabolic studies revealed that the CMS plants had an abnormal starch accumulation with a concomitant reduction of sucrose levels when compared to B. napus. The CMS plants are mainly characterized by the presence of carpelloid structures in the third whorl of the flower, replacing the stamens of B. napus, and by petals reduced in size. Histological and ultrastructural studies made of young flower buds showed that the cell division pattern in the putative whorls two and three was altered. Cells in the CMS lines had divided in several directions instead of the typical anticlinal cell division present in the two first layers of the B. napus young flower bud. The same alterations in cell division patterning were also observed in the vegetative meristem of the CMS lines. In both these two meristematic tissues (vegetative and floral), two mitochondrial populations were found in the CMS lines. One population of mitochondria resembled the B. napus ones at the ultrastructural level but were always smaller in size. The other population showed disrupted inner-membrane systems and the density of the matrix was strongly reduced. In accordance with the disrupted mitochondria, flower tissues from the CMS plants displayed reduced levels of ATP in comparison to B. napus. All the homeotic genes and their upstream genes showed the same expression pattern in young flower buds between the three lines until third whorl organs had differentiated. Even though the pattern was similar, the expression levels of the same genes showed differences between the two CMS lines and B. napus. By the time third whorl organs started to differentiate, BnAP3 and BnPI expression levels were strongly reduced while the upstream genes like BnUFO and BnLFY were up-regulated. This up-regulation suggest that the action of BnUFO and BnLFY is interrupted when activating BnAP3 and BnPI that in turn, seems to reflect a feedback up-regulation mechanism by the nucleus. This feedback regulation can be explained by the fact that the nuclear genes in the CMS cells are not mutated. The transcription of nuclear genes coding for non-functional proteins will then be up-regulated. The hypothesis developed in this study relates the reduced levels of ATP in the flower tissues of the CMS lines with the protein degradation of key proteins necessary for correct flower development and cell division.
Plant cell reports, 2011
In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A ‘mild’ heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3–8 celled.
Cytoplasmic male sterile line RC7 of Chinese cabbage produces mature anthers without pollen. To understand the mechanisms involved, we examined the ultrastructural changes during development of the microspores. Development of microspores was not affected at the early tetrad stage. During the ring-vacuolated period, some large vacuoles appeared in the tapetum cells, making them larger, extending to the anther sac center during the monocyte period. At the same time, the tapetum degenerated as the microspores aborted, resulting in pollen-deficient anthers. As a result, the locules collapsed and the anthers shriveled. The callose was degraded in the pollen walls; abnormal deposits of electrodense material gave rise to irregular spike-shaped structures, rather than the characteristic rod-like shape of the B7 bacula. The internal intine wall of RC7 was thinner than that of the B7 type. At the mitosis I microspore stage, the tapetum cells contained multiple plastids, with numerous small spherical plastoglobuli, and lipid bodies. Based on these observations, we suggest that RC7 abortion may be due to mutated genes that normally regulate development of the pollen wall and cell walls in the RC7 line.