The Cytology Study On The Free Nuclei Mitosis Of Female Gametophyte In Pinus Tabulaeformis Carr.

The cones of clone 28 can aggrandize normally, but their ovules aborted due to the free nuclei mitosis failure of female gametophyte in the seed orchard of Pinus tabulaeformis Carr. in Xingcheng, Liaoning Province. It is an excellent material for mechanism research on the female gametophyte development of woody plants with its steady genetic characteristic of female sterility. The projective of this study was to analyze the mechanism of the free nuclei mitosis failure in this female sterility clone. After system studies of cytomorphology and histochemistry in ovules during the free nuclei mitosis failure, a fine cell culture from the embryos of P. tabulaeformis was established in this paper. The regulation mechanism of cell cycle in P. tabulaeformis was analyzed by cell synchronization, flow cytometric and 2-dimensional electrophores. Western blotting and immunohistochemical were applied to exploring the expression of the cell-cycle regulating factors during the free nuclei mitosis failure. The results were valuable information for understanding the expression and regulation of cell-cycle associated factors during female gametophyte development of gymnosperm, which were significant for extending study field of woody plants developmental biology and uncovering the mechanism of female sterility.1. The changes of the microstructure, ultrastructure and nutrient in the integument, female gametophyte and free nuclei in the clone 28 and clone 11 during the free nuclei mitosis failure were studied by isolation technique of female gametophyte, electron transmission microscope and histochemistry. The female gametophyte development in the female-sterile clone stopped at the stage of free nuclei. The cell organs of female gametophyte in the female-sterile clone were abnormal, the nucleus became swollen,the nucleolus disappeared, and the chromatin began to condense. There were large amounts of abnormal mitochondria swelling and extending in the cytoplasm of female gametophyte and their inside structures became unclean, while the number of vacuole decreased significantly even no larger vacuoles were found in the cytoplasm. Depression and rupture appeared in tonoplast. Cell walls were getting thick in nucellus cells near the female gametophyte and polysaccharides accumulated abnormally.2. The result of study on the cell cycle in P. tabulaeformis showed that suspension cells in exponential phase treated with 3 mM HU for 24h, after subcultured for 3h following the treatment, 90.71% cells were in phase G1; cultured for 27h after the removal, about 40% cells entered in phase G2, meanwhile the maximum number of cells in metaphase/anaphase was found. The changes of protein components between cells in 3h and 27h after the removal were analyzed by 2-dimensional electrophoresis. The result showed there were 11 proteins expressed only in phase G1 and 4 proteins expressed only in phase M. The volume of 7 proteins increased and the volume of 3 proteins decreased in phase M compared to proteins of cells in phase G1. These proteins distributed mainly in 18kDa/pI6.3-30kDa/pI7.5 and 60kDa/pI6.5-70kDa/pI7.2, and may be necessary for regulation of distinct phases in cell cycle.3. It was found that CaM and Cyclin C-like protein played an important role in the free nuclei mitosis of female gametophyte. The result showed that the expression of CaM and Cyclin C-like protein changed similarly throughout the cell cycle, the content of CaM and Cyclin C-like protein in phase G1 was little, and increased greatly in phase S, then in phase G2 the content decreased. After that, cells entered into next cell cycle, the content increased again in phase S. These results indicated that CaM and Cyclin C-like protein involved in the cell cycle regulation of P. tabulaeformis. The distribution of CaM,Cyclin C-like protein expressed the time-space-regulation specificity. At the stage of free nuclei vigorous division, the CaM distribution in the layers of big cells in integument decreased, however, the CaM distribution in the outboard cells in the integument increased obviously. Therefore, the integument was probably taken as a synthetic site or a accumulated and scattered site of CaM, and regulated free nuclei mitosis and cell degradation in nucellus. The content increase of CaM in free nuclei suggested to be related to cell wall formation during cell generating process in female gametophyte. Cyclin C-like protein expressed following free nuclei vigorous division. Overexpression of Cyclin C-like protein at the late stage of the abortion suggested it might involve in the regulation of free nuclei mitosis, and its aberrant expression may be an important factor influencing the female gametophyte fertility.