Induction of embryogenesis in [isolated] microspores and pollen of Brassica napus L. cv. Topas

B. Hause, G. Hause

Research output: Thesisinternal PhD, WU


<br/>Artificial systems to produce plant embryos are important tools for basic research as well as for plant breeding. It is possible to produce large amounts of embryos by methods like somatic embryogenesis or embryogenic microspore cultures. Such high amounts of embryos, which are easier to handle than zygotic embryos, are the prerequisite for biochemical and molecular genetic investigations on the one hand, and for biotechnological use on the other hand. Moreover, embryos derived from microspores or pollen represent a very efficient basis for the production of plant hybrids: Because of their haploid origin, microspore-derived embryos are after diploidization dihaploid, and thus, homozygous.<p>The induction of embryogenesis in microspores and pollen of <em>Brassica napus is</em> realized by their cultivation under heat shock conditions (32 °C for at least 24 h). This dissertation presents results from our investigations on the induction phase of embryogenesis. Cellular changes during the first 24 h of cultivation of microspores and pollen were analysed to discern cytological differences between this induction phase of embryogenesis and the normal pattern of pollen development <em>in planta.</em> Combining light as well as electron-microscopy for the analysis of the cytoskeleton (microtubules and microfilaments), it was shown that the development of microspores and pollen in <em>B. napus</em> to a mature, tricellular microgametophyte is comparable to other angiosperms. Only the disappearance of the central vacuole before microspore mitosis is different from the common pathway (Chapter 2). Stage specific developmental patterns of microtubules and microfilaments could be detected in the microspores and pollen.<p>Symmetrical divisions are a prerequisite for the embryogenic development of cultivated microspores and pollen, and three pathways for the induction of such divisions were identified (Chapter 3). (i) In vacuolated microspores cultivation under embryogenic conditions causes a migration of the nucleus to the centre of the cell where the division takes place. This phenomenon is accompanied by the disruption or altered formation of the microtubules. (ii) In late microspores the embryogenic conditions cause a turn of the mitotic spindle up to 90°. Both events (i and ii) result in symmetrically divided cells forming a bicellular proembryo. (iii) The developmental switch from young, bicellular pollen to the formation of proembryos is caused by a disrupted pollen development (arrest of the generative cell at the pollen wall) followed by division of the vegetative cell. In this case, microtubules, normally detectable in the vegetative cell perpendicular to the generative cell, are disrupted. In summary, all three pathways can lead to the formation of proembryos, and the microtubular cytoskeleton seems to be involved in these developmental changes. Although changes were also visible concerning the microfilaments under embryogenic conditions, their role in the induction of embryogenesis could not be confirmed.<p>Because of the obvious changes in the cell cycle of microspores and pollen cultivated under embryogenic conditions, the synthesis of DNA was investigated <em>in vivo</em> and <em>in vitro</em> (Chapter 4). The incorporation and detection of bromodeoxyuridine as well as the determination of the ploidy level of the nuclei by microspectrophotometry were used for these investigations. DNA replication could be shown <em>in vivo</em> within the nucleus of the late microspore and also within the generative nucleus of the late bicellular pollen. In normal development the vegetative nucleus remains in the G1- phase. Under embryogenic conditions, the pattern of replication in microspores remained the same, but the vegetative nucleus of the young bicellular pollen re-entered the cell cycle and exhibited DNA synthesis.<p>Changes in phosphorylation patterns were analysed using the monoclonal antibody MPM-2 (Chapter 5). The antibody MPM-2, raised against mitotic proteins of HeLa-cells, recognizes phosphorylated, mitosis-specific proteins in animal and plant cells. In developing microspores and pollen of <em>B. napus,</em> MPM-2 bound to proteins of all developmental stages, especially to proteins in the nuclei. Moreover, there were no differences in phosphorylated epitopes between microspores and pollen cultivated under embryogenic and non-embryogenic conditions. This might be caused by the fact that this antibody recognizes phosphorylated epitopes of various proteins.<p>Because of the heat shock conditions used for the induction of embryogenesis, the subcellular localisation of heat shock proteins (HSPs) was performed (Chapter 6). Western blot analysis of proteins separated by two-dimensional gel electrophoresis revealed a strong signal at 70 kDa. Immunocytochemical investigations using an antibody raised against HSP70 showed a distinct stage- specific subcellular localization of HSP70 <em>in vivo</em> as well as <em>in vitro.</em> The embryogenic cultivation caused an altered localization of HSP70, which became detectable within the nucleus of the vegetative cell. Its localization could therefore be correlated with the initiation of DNA replication. Possible relations between HSP70 and replication were discussed.<p>Chapter 7 describes the localisation of a specific mRNA within developing microspores and pollen of <em>B. napus</em> and <em>Arabidopsis thaliana.</em> Using freeze sectioned material and <em>in situ-</em> hybridization with a digoxygenin labelled probe, specific gene expression was demonstrated for the generative cell of both species.<p>Finally, the expression of polarity during the development of microspore-derived and zygotic embryos was compared (Chapter 8). Investigations by scanning electron microscopic techniques showed that the embryo formation between them are similar from the globular stage onwards. The distribution of calcium ions, calmodulin and starch was used to find early signs of polarity. However, the accumulation of starch and the position of a residual pollen wall were the only hints for a predisposition of the radial axis of the developing embryo.<p>In chapter 9 the embryogenesis in isolated microspores and pollen is considered as a biphasic process. The induction phase of embryogenesis represents the dedifferentiation of a developing organism followed by differentiation to a real plant embryo. Our results are combined with results of other groups to create a general scheme on induction of embryogenesis in microspores and pollen of <em>B. napus.</em>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Willemse, M.T.M., Promotor
  • van Lammeren, A.A.M., Promotor, External person
Award date19 Nov 1996
Place of PublicationS.l.
Print ISBNs9789054856146
Publication statusPublished - 1996


  • plants
  • embryology
  • reproduction
  • tissue culture
  • cell culture
  • meristems
  • brassica napus var. napobrassica
  • swedes
  • embryo culture

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