Calcium Location Of Anther And Exploring Of Fluorescence Labeling Methods For Cytoskeleton Of Pollen Of Rice

Photo-sensitive Genic Male-sterile Rice(PGMR) is a very important germplasm resource for cross-breeding of rice. Its cytological mechanism of male sterility including many aspects: the development of tapetum, ATPase, concentration of Ca²⁺, cytoskeleton and programmed cell death, etc. Ca²⁺widely participate and regulate physiological and biochemical response in plants as second messengers, its very important for the formation and development of pollen during the development of anther; disorder of cytoskeleton lead to cell death is general. Therefore, researching the relationship between pollen abortion both distribution of Ca²⁺ and variation of cytoskeleton during the development of anther of rice, helpful to reveals the mechanism of male sterility for PGMR.Potassium antimontate was adopted to investigate Ca²⁺ distribution in the anthers of photo-sensitive genic male-sterile line of rice Nongken 58S and fertile line Nongken 58N during their development, to study the relationship between Ca²⁺ distribution and male-sterile of rice. In order to investigate the relationship between the dynamic distribution of cytoskeleton and male-sterile of rice, a feasible immunofluorescence labeling method must be established firstly to observe the framework of cytoskeleton of pollen. We attempted several ways to gain pollen and made great efforts to improve experimental method, finally, a mature labeling system was established in our laboratory, by which we can label both microfilament and microtubule easily.The main results are listed as bellows:1. To research the relationship between Ca²⁺ distribution and male-sterile of rice(1) In the locule of fertile anther, the calcium precipitates gradually accumulated on the surface of uninucleate pollen until pollen mature, but its lack in the cytoplasm, the pollen wall formed completely composing of exine and intine. The calcium precipitates on the surface of sterile pollen were obviously fewer than fertile pollen, but they were abundant in the degradated cytoplasm of abortive pollen, the exine formed singularly and the pollen wall composed without intine.(2) The tapetum cells of the fertile anther began to degenerate at early microspore stage and degraded rapidly at late microspore stage, but that of sterile anther startuped degenerate at microsporocyte stage, and went on to pollen abortion. Ubish bodies began to form earlier in sterile anther than fertile anther, but with no calcium granules on them, till late microspore stage, many calcium granules appeared on the surface of them, but obviously less than in fertile anther. In sum, there were much more calcium granules in anther wall of sterile anther than in fertile anther, but the calcium granules on the surface of ubish bodies in sterile anther were much less than in fertile anther.(3) The calcium precipitates gradually increase in parechymatous cells of both sterile and fertile anther, but there were more in sterile anther than in fertile anther.Due to the tapetum cells degrading ahead and the abnormal function of ubish bodies, the transport of Ca²⁺ to locule occurred hindrance, induced to form abnormal exine. Abundent Ca²⁺ in the cytoplasm was one of the most factors of the pollen abortion.2. A labeling system was established to observe the cytoskeleton of pollen.(1) Pollen were extruded out from anther and were allowed to settle on the coverslip. The anther wall debris was removed, but difficult to clean out fully. The pollen lose easily during the process of experiment. The anther wall debris disturbed the backdrop of fluorescence. The method was not adapt to labell microtubule easily.(2) Gain pollen through grinding and centrifugation. This was used successfully to labell both the microfilament and microtubule. During the process of experiment, only a few pollen lost, and the backdrop of fluorescence was very clean. But it was difficult to gain the early pollen.(3) Used freezing apparatus to slice anther to flake could obtain pollen of each stage. its was easily to slice but was difficult to labell the cytoskeleton of pollen.(4) The pollen under label were photographed under both fluorescence microscope and Confocal microscope. The results showed that the cytoskeleton under the Confocal microscope was clearer and more bright than under the fluorescence microscope.It was obvious that these methods have virtue as well as defect. We could used two methods simultaneously to labell the pollen during its development. But it was a pity that we fail to label pollen by slicing.