Experimental Study On Early Embryogenesis

In angiosperm, double fertilization is followed by embryogenesis and endosperm formation. The maternal-to-zygotic transition (MZT) is a major developmental switch during early embryogenesis. In animals, it is clear that there is a delay between fertilization and the maternal-to-zygotic transition and early embryogenesis is largely controlled by maternal transcripts deposited in the egg cell before fertilization. However, in higher plants little is known about the MZT. When it occurs and what its biological impact is in embryogenesis are still hardly understood. Based on in vitro and semi-in vivo embryogenesis systems, we employed transcription inhibitors to block de novo transcription in zygotes at different developmental stages in order to study the role of MZT during embryogenesis. The main results are summarized as follows:1. The fertilization occurs at about 48 HAP (hours after pollination). There is 2-3 day interval from fertilization to the zygote first division in tobacco. During this period several morphological changes take place. The zygotes of tobacco will go through shrinkage, directional elongation along the longitudinal axis which accompanied with the polarity reestablishment. The time course of fertilization and morphological changes were confirmed by artificial pollination together with the observation of fixed embryo sacs and isolated zygotes. The data offered the standard for material selection and morphological comparison in following study.2. There are three candidate concentrations of the inhibitions for selection. The chosen concentration could efficiently block the growth of cells, which were tested by both ovule culture and leaf protoplasts culture system. Furthermore, RT-PCR test revealed that the transcription inhibitors indeed blocked the gene activation in zygotes.3. We set up an improved the in vivo-in vitro ovary culture system that can guarantee both embryogenesis efficiency and convenient inhibitor treatment. Based on the systems, the unfertilized but pollinated ovaries were treated by the inhibitors to make sure total inhibition of the de novo transcription after fertilization. It was found that the inhibitor treatment made the zygote development arrest at the early stage, similar to 72HAP zygotes in vivo, and the first division was totally blocked. The transcription inhibitor disturbed the development of the zygotes seriously. It indicates that the maternal factors can support very limited development after fertilization and the MZT initiates at early stage before the zygotic first division.4. We employed the fertilized ovaries at 67HAP and 72HAP for further transcription inhibition to testify the occurrence and importance of MZT during early embryogenesis. In 67HAP ovaries treated by the inhibitor, the zygote arrested at the similar stage to that in unfertilized ovaries. Also, in 72HAP zygote culture, the zygote can’t divide. These results indicate that the MZT initiated before the first division and the de novo transcription is essential for the zygote later development and zygotic cell division.5. Based on the understanding of the MZT initiated before the first division, we further treated the 96HAP zygotes. The frequency of the first division about 90% in zygote culture without inhibitor treatment. However, after inhibitor treatment, ratio of division was only about 50%. Furthermore, in cultured ovaries at the corresponding stage (91 HAP), the embryos could grow to the 4-cell stage after 53 hours inhibitor treatment, at the meanwhile, in the control embryos could grow to 8-cell stage. It indicates that the MZT initiated during 72 to 96HAP and the de novo transcription already existed in the 96HAP zygote, which can support the zygote divide once at least. And the further treatment on the 2-celled proembryo showed that the embryos could develop to the 8-cell stage. This suggests that the de novo transcription or translation of them is a successively progressed.6. When transcription inhibitors were used on the zygote before 72HAP, the development of zygotes was very limited. The large vacuole disappeared and the cell volume shrank as expected. But the cell wall synthesis, the elongation and the first division of the zygote were blocked. It indicates that the maternal factors can only support the egg cell transforming to a zygote. When transcription inhibitors were used on the zygote at 96HAP, the cell wall synthesis shows no obvious difference and the cell division was not blocked. It suggests that the zygotes later than 96HAP have already been 1-cell embryo. Therefore, we suggest that egg cell development after fertilization can be divided into two stages:zygote and one-cell embryo stages.7. In the observation on the vesicles stained with FM4-64, it was found that the inhibitor treatment has no obvious influence on endocytosis. And the cyclosis is also active. These results suggest that the zygotes are living and basis vital movements are normal. And after the inhibitors cleared, the ovules and zygotes in cultured ovary have limited degrees of growth. In whole mount clearing ovules and the isolated fixed embryo sacs, the elongated zygotes were observed. But there is no further stages of embryos were found. Thus, the developmentally blocked zygotes by inhibitor treatment are unable to recover. This suggests that the MZT occurs in an exactly preprogrammed time course and ordering degradation and generation of its transcripts;once the time course and the order are disturbed they are hardly re-established.