Determination Of The Apical Cell Developmental Fate In Relation To Apical-basal Cell Interaction In Nicotiana Tabacum L.

As one product of double fertilization in higher plants, zygote is the starting point of plant sporophyte generation. The zygote experiences cell division, differentiation in order to complete embryogenesis and form a mature embryo, ultimately, become the important component of seeds. Plant embryogenesis is a very complicated process including a series of choiceness and orderly developmental events. Two different daughter cells emerge after the asymmetric cell division of zygote. One cell located in micropyle end of ovule which was called basal cell, while the other located in chalaza end of ovule which was called apical cell. Apical cell is smaller, containing thick cytoplasm and rich organelles; basal cell is larger,containing a big vacuole. The differences between apical and basal cell are caused by the polarity and asymmetric cell division of zygote. Apical cell becomes the predecessor of embryo proper, while basal cell becomes suspensor after limited horizontal cell division. Therefore, the cell structure and cell fate between apical and basal cells present markedly different. Apical and basal cells derived from the zygote, but apical and basal cell fates are different. When and how are the apical and basal cell fates determined? Is there any cell-cell interaction between apical and basal cell? However, there are no perfect answers yet for such problems.In the present study, we try to follow the development result of apical cell without basal cell via laser cutting technology and the tobacco in vitro micro-culture method. We concentrate the apical cell fate and its relationship with the cell-cell interaction between apical and basal cells, and we hope to get definite understanding of the time of apical cell fate determination and its relation to basal cell and suspensor development. The experimental results are as follows:1. We obtained the precise division pattern from zygote to early globular embryo stage through manual microscopic operation and transparent observation methods. This provided a direct evidence for our later study on embryogenesis. Comparison of above two kinds of observation methods, we can confirm that apical cell, as one of daughter cell from zygote firstly experiences horizontal division which was perpendicular to apical-basal axial, followed by two mutually perpendicular longitudinal division to form early globular embryos. In the process of early embryogenesis, we deeply observed important characteristics of embryonic cell division and development pattern which provides a comparative mode for study on embryogenesis in vitro.2. Combined with the established culture system in vitro with tobacco zygotes, we further improved in vitro culture conditions for2-celled proembryo. The experimental results indicated that the added spermindine could promote cell division and embryogenesis in vitro. The above conditions for in vitro culture conditions with2-celled proembryo provided a good technology foundation for our research on cell fate determination of apical and basal cell.3. We collected many apical cell via the successfully applied method in plant——laser control microdissection(LCM). We also testified and validated the vitality of these cells. According to different cutting technology, we obtained three types of apical cells and cultured them in vitro. After observation on the development result of three different apical cell cultured in vitro, we found that the apical cells with parial basal cell residue can maintain the continued cell development which were good for our research on apical cell. Those cells provided us valuable material.4. We found that the independent apical cells can divided to form a young embryo with4-8cell via continued observations and detailed records of all apical cells cultured in vitro and comparison with normal developmental pattern.63.6%of apical cells carried out the same first division in vitro as in vivo, it was transverse division. Further more,26.8%independent apical cell could make at least three cell division and form a young embryo in vitro, while68.5%normal apical cell could form a young embryo after three cell division. Above data suggested that lacking basal cell does not affect apical cell fate in vitro, but its continued development in vitro without basal cells is restrained. In addition, basal cell also influenced the cell division pattern of some cells which was at the basal end of apical cell products.5. We proved that the apical cell product was embryonic after RT-PCR detection using contig824as a embryonic gene maker. Using the early embryos specifically expressed marker gene DRP (NtDRP::GFP), we observed its expression pattern in apical cells in vitro culture products. We also confirmed that the apical cells in vitro culture products had embryonic characteristics.6. Combined with the apical, basal cells development results from culture in vitro, we want to detection material transport between apical cell and basal cell via tracking photo-activated green fluorescent protein(PAGFP) transport between above two cells. We constructed ProNtDRP::PA GFP vector, and transformed it into tobacco. We acquired positive transgenic plants through the PCR detection and fluorescence observation. We have groped suitable conditions for the operation and observation on the distribution of PAGFP in early embryo including2-celled proembyro. By comparing PAGFP excitation light intensity, activation patterns, activation area and so on, we found that the following conditions such as50%excitation light intensity, using a point activation patterns, activation in the nuclear, not cytoplasm were more suitable for observed fluorescent protein transport.7. After observing PAGFP in2-celled proembryos, we traced transport of PAGFP between apical and basal cells. We found that GFP in15.8%activated basal cell could transport from basal cell to apical cell after activation, whereas GFP in all activated apical cell in experiments did not transport from apical cell to basal cells. It suggested that some substance like GFP transport between apical cell and basal cell was selective, in which the transport from apical cell to basal cell was harder and from basal cell to apical cell. Maybe the polar transport between apical cell and basal cell affects apical cell sustained development. 8. We found that the destroyed suspensor could quickly recover after cutting the uppermost cell in suspensor utilizing LCM technology. The recovered suspensor also grew normally and formed a integrated suspensor. It indicated that the suspensor had a strong ability of self-repair. On the basis of unaffected embryo proper development, it indirectly demonstrated that embryo proper development was dependent on suspensor development.In summary, we found a small effective molecule——spermindine which can promote cell division and in vitro embryogenesis making use of in vitro single-celled micro-culture. On above basis, combining the embryonic development specific gene expression detection and special photo-activated green fluorescent protein observation technology, we traced the apical cell development by continued observation. Our experiment results indicated that apical cell fate was not affected by basal cell in vitro, but its continued development in vitro was affected by basal cells, the polar transport between apical cell and basal cell maybe resulted in the affected apical cell development. All the experiment data above were good for us to understand that when and how the apical cell fate is determined and whether there is any cell-cell interaction between apical and basal cell.