Cytological Study And PCD Assay On Pollen Development Of Photoperiod Sensitive Genic Male Sterile Rice

The photoperiod sensitive genic male sterile (PSGMS) rice was discovered as aspontaneous mutant from a japonica rice (Oryza sativa L. ssp. japonica) cultivar Nongken58 (abbreviated as 58N). A major characteristic of the PSGMS rice (referred to as 58S) isthat its pollen fertility is regulated by day-length: the pollen is completely sterile underlong-day conditions, while the fertility varies from partial to fully fertile under short-dayconditions. This characteristic makes it a desirable germplasm for developing "two-line"hybrids in rice breeding programs. The PSGMS rice has also provided a very interestingmaterial for investigating male sterility in plants. Therefore, it is necessary foridentification of the cytological cause for the abortive development. Despite itswidespread utilization in breeding programs, the precise initial stage of the abortivedevelopment of the PSGMS rice and the possible cytological mechanism of pollenabortion have not been determined. In this study, we conducted a systematic cytologicalobservation of the anthers of the PSGMS rice 58S in comparison with its fertilecounterpart 58N. A TUNEL assay was also performed comparatively to reveal DNAfragmentation in the anthers of both 58S and 58N. The objective was to identify, at thecytological level, the causal mechanism for the male sterility in the PSGMS rice.The main results are as the follows:1. No difference were observed between 58N and 58S (under natural short-dayconditions) in anther and pollen development processes. The anther wall differentiatesinto 4 layers at sporogenous cell stage. The degeneration of tapetal cells initiated atuninucleate microspore stage and completely disintegrated at the bicellular pollen stage.After releasing from the tetrad, the separated cells develop into microspores. Themicrospores underwent two consecutive mitotic divisions led to a tricellular pollen grain,also referred to as mature pollen. Few anthers had six pollen sac or two vascular bundles.2. Under natural long-day conditions, the results showed that pollen abortion in thePSGMS rice first occurred no later than the pollen mother cell (PMC) stage and continuedon in the entire process of pollen development till pollen degradation. This abortive process was closely associated with the abnormal behavior of the tapetum. Althoughtapetum degeneration in the PSGMS rice initiated at as early as the PMC stage, itproceeded slowly and did not complete until the breakdown of the pollen, which is insharp contrast to the rapid disintegration of the tapetal layer during late microspore tobicellular pollen stage in the fertile rice.Base on abundant observations, we examined abnormal anther numbers from thePSGMS rice at different stages of pollen development. The abnormality occurred in thetapetal cells as evidenced by condensation and vacuolation of the cytoplasm, which wereobserved in approximately 65% of anther examined from 58S. While the majority ofPMCs could complete meiosis in the PSGMS rice 58S, abnormality was observed inabout 19% of the anther observed.3. Such cytological observation was supported by the results of the TUNEL assaythat detects DNA fragmentation resulting from programmed cell death (PCD), indicatingthat the tapetum degeneration is in the process of PCD. At the PMC meiosis stage, thenuclei of tapetal cells in anthers from 58S showed intense yellow-green stain, whereas nopositive signal was detected in anthers from 58N, suggesting that molecular events ofPCD in 58S had already initiated at this stage. The results demonstrated that thepremature PCD of tapetum was the main cause for microspore abortion.4. Our study found the correspondence relation of the length of anther and the pollendevelopmental stages, which provided morphological references for sampling in the field.