Cytological Observations Of Programmed Cell Death In Anther Of Cytoplasm Male-Sterile Pepper(Capsicum Annum L.)

Pepper(Capsicum annum L.) is a kind of very important vegetable crops with a high nutritional value and economic value and also has a significant heterosis. Cytoplasmic Male Sterility(CMS) is the foundation of pepper heterosis utilization and is closely connected with Programmed Cell Death(PCD). In order to determine the cytological mechanism of pollen sterility in CMS pepper, it was carried out using pepper male sterile line 8214 A and its corresponding maintainer line 8214 B as materials, to observe the positive TUNEL(the method of terminal DNA transferase mediated Nick end labeling) signals in the microspore mother cells and the tapetal cells of sterile lines and maintainer lines by using detection technology of TUNEL to understand the occurrence of nucleus DNA breaks, and as well as the changes of the characteristics of the Ca2+ distribution in the microspore mother cells and the tapetal cells of the sterile lines and maintainer lines in abortive process by using transmission electron microscopy.The results showed that the nuclei of microscopes mother cells(MMCs) in sterile lines had obvious positive signals at the beginning of the meiosis stage and being proved to be fragmentation of nuclear DNA by TUNEL detection. The nuclear DNA of MMCs in the male sterile line was common during meiosis stage, and that was not the same as in the maintainer line. At tetrad stage, the nuclei of the tapetum cells both in the male sterile line and the maintainer line were TUNEL positive reaction, which showed the tapetum cells appeared the programmed cell death.The results of ultracytochemical localization of calcium showed that the nuclei of MMCs in the maintainer line are large and located in the middle of cells and the anther wall cells have completely differed just before meiosis of MMCst. At this time, a few calcium particles were mainly distributed in the vacuole of cytoplasm and the wall in MMCs, and as well as the wall of tapetum cells. At early meiosis stage, MMCs were enlarged with dense cytoplasm and appeared clear visual chromosomes with nuclei being disappeared. The granules of calcium in MMCs were gathered in the small vacuole of cytoplasm, The tapetum cells in anther wall were radially enlarged and the number of organelles also increased. Some granules of calcium in the tapetum cells were gathered in the walls and the intercellular. At the end first division of the meiosis stage, there appeared two nuclei in the MMCs and some calcium granules were gathered in the callose wall. At tetrad stage, four microspore cell in tetrad were ranked tetrahedron and surround by the common callose wall, which had dense cytoplasm, and began to form the extin where were gathered many calcium particles. At this time, the tapetum cells appeared big vacuole and began to become irregular shape and many calcium particles were distributed in the wall and intercellular gap of cells. At the early microspore stage, the nuclei of microspores were big and located in the center of the cells, and many calcium particles were uniformly distributed in the extin. The tapetum cells began to show signs of degradation and the distribution of calcium was the same as that before stage. At the late microspore stage, the microspores appeared a big vacuole, and the extin were completely formed where many calcium granules were gathered. The tapetal cells were completely degraded and many calcium granules were gathered in the existing remnants. When microspores completed the first mitosis and formed bicellular pollen containing a large vegetative cell and a small reproductive cells. At the mature pollen stage, the pollens were accumulated a large number of starch grains in the cytoplasm. Many calcium granules were gathered in the extin of pollens during bicellular and mature pollen stage. The anther wall at mature stage only existed the epidermal cells and endothecium cell with fibrous stripes where were uniformly distributed with some calcium particles.MMCs and anthers wall cells in sterile lines were similar to those in maintain line before meiosis stage. Then the cytoplasm of MMCs appeared local contraction after the first division of meiosis stage. At the same time, the tapetum cells in anther walls showed the sign of degradation with irregular cell shape and incomplete nuclei. At this time MMCs in sterile line did not appear the same distribution characteristics of calcium particles as those in maintainer lines, which had few calcium particles in the former. It was observed that the anther locui was not enlarged, and abnormal tetrad without common callose walls being adhered each other which the number of microscopes was uncertain and the size of microscopes were the same in the tetrad after meiosis. The calcium granules were still few in the tetrad. At this time, the tapetum appeared to degrade after meiosis and many calcium granules were gathered in the intercellular gap and inside tangential wall of degenerating tapetum cells. Then, the cytoplasm in the tetrad microspore further contracted and completely degraded and some calcium granules were gathered in the degraded remnants. There were no pollen in the mature anther.Therefore, the pollen abortion in the CMS pepper was associated with the PCD of MMCs and decreases of calcium ion of MMCs during meiosis might closely be related with its own PCD.