Isolation Of The Sperm, Egg, Central Cell, Synergid Cells And Proembryo Cells Of Torenia Fournieri

Our understanding of fertilization mechanism of higher plants is very limited because of their complexity of the reproductive structure in which the male and female gametes are embedded deeply in various somatic tissues. Following the successful isolation of male and female gametes from higher plants, the in vitro fertilization can be conducted, which makes it possible to study the fertilization mechanism without the influence of somatic ovary and ovule tissue. Isolation of egg cell, central cell, synergid cell, zygote, and proembryo also makes it possible to study the functions and structures of these cells by using modern cytological and molecular methods. Torenia fournieri is a good model plant for studying its fertilization biology because its embryo sac partially protrudes out the micropyle, and its egg cell, two synergids and part of the central cell can be clearly observed by using light microscope. In present studies, we were able to successfully isolate sperm cells, mature and living egg cells, zygotes, central cells, synergid cells, apical cells and basal cells from proembryo of Torenia fournieri, which makes the basis for in vitro fertilization of Torenia fournieri, and opens an access to use molecular methods to study fertilization mechanism of Torenia fournieri.1. Isolation of sperm cells: The pollen of T. fournieri is bicellular type, containing a generative cell and a vegetative cell in a pollen grain at anthesis. The division of the generative cell and the formation of two sperm cells of T. fournieri occur in a growing pollen tube within the style at 2 h after pollination. We used a semi-in vitro technique in which the stigma was pollinated and the pollen tubes grew in vivo for 5 h, then the style was excised and immersed into culture solution. When pollen tubes grew out of the cut end of the style after 2-3 h, a pair of sperm cells could be released by bursting the pollen tube. A pair of the sperm cells of T. fournieri displayed a significant difference (P<0.01) in size. The two brother sperm cells of T. fournieri are dimorphic cells and also displayed viability differences as indicated by fluorescence of fluorescein diacetate (FDA) staining. The fluorescence of the sperm associated with the vegetative nucleus (Svn) was brighter than the sperm unassociated with the vegetative nucleus (Sua). The collection of the two individual populations containing over thousands cells will permit to cDNA libraries construction of both sperm cells which will help us to understand the differences at the molecular level.2. Isolation of the egg cells and zygotes: After the observations of pollen tube growth and stages of embryo sac of T. fournieri, we were able to successfully isolate a large number of living egg cells and zygotes. The cytological changes of egg cell and synergid cells before and after fertilization are very evident and easily to be identified. We observed a starch strand formed within ovules short after the pollen tube entering embryo sac, and no starch strand was formed in unfertilized ovules, suggesting that the changes of nutriment in the ovule is induced by fertilization. The size of the isolated egg cells changed after fertilization and zygotes became smaller. The surface charge and electrophoretic mobility between egg cells and zygotes were compared by using micro-electrophoresis. When egg cells and zygotes were put into an electron field filled with optimal buffer, they both migrated toward negative pole, indicating that both egg cells and zygotes have positive charge on the cell surface. Zygotes moved quicker than egg cells, suggesting the surface charges of the zygote increased after fertilization. The isolation of vigorous egg cells and zygotes provides an opportunity to study fertilization mechanism.3. Isolation of the central cells and synergids: Unfertilized and fertilized central cells and synergid cells of the different stages were also isolated by improving isolation techniques. Central cells are of significant differences in size and at states before and after fertilization. An unfertilized central cell, which contains many large vacuoles and less starch grains, is smaller than a fertilized central cell, in which the large vacuoles disappeared, and starch grains are full of the cell. We isolated the synergid cells of anthesis and 2d after anthesis and the different stages of synergid cells are of a little difference in size.4. Isolation of the proembryo cells: Fertilization leads to a remarkable biologic phenomenon: the process of embryogenesis. After fertilization, zygotes start to divide and progeny cells undergo a series of division and differentiation steps, finally generate a new sporophyte--embryo. The molecular mechanisms of early embryogenesis have been most intensively studied in animals. However, little is known about activation of the zygotic genome and early embryogenesis in angiosperms. With the advance of researches of fertilization mechanism and embryogenesis in higher plants, the mechanism of early embryogenesis attracts great interests from many scientists. The differentiation mechanism of the apical cell and basal cell of a bicellular proembryo is a very interesting topic in plant developmental biology, but we know little about it because it is necessary to isolate the apical cell and basal cell of a bicelluar proembryo first. We isolated some apical cells and basal cells of bicellular proembryo of T. fournieri, which make the basis for comparing the cDNA libraries of zygote with that of the apical cell and basal cell to investigate the differentiation mechanism of apical cell and basal cell at the molecular level.