| The technology of crossbreeding has greatly improved the yield of rice.It played an important role in ensuring living standard and food security.The cultivation methods of hybrid rice in our country mainly include three-line hybrid system and two-line hybrid system.Photoperiod-/thermo-sensitive genic male sterility(P/TGMS)is essential for two-line breeding systems,which exhibits male sterility at high temperature and restored fertility at low temperature.Compared with the cytoplasmic male sterility of three-line breeding systems,the P/TGMS of two-line breeding system has a broader range of germplasm resources,which has been successfully applied in agriculture.However,with the increasing occurrence of extreme weather,P/TGMS lines was hard to maintain the stable sterility and reduce the purity of hybrid seed production.Therefore,the identification of new P/TGMS lines is important for promoting the development of two-line hybrid system.Our previous studies proposed a general fertility recovery mechanism of thermo-sensitive male-sterility plants.In this study,we found a new thermo-sensitive male-sterile mutant k18 by EMS(Ethyl Methane Sulfonate)screening,which showed the phenotype of high temperature sterility and low temperature restores its fertility.The pistil of k18 have the normal function by crossings experiment.In Shanghai,2020,the statistic analysis showed that the sterile rate and recovery effect of k18 were comparable to those of tms5.Moreover,in 2021,due to the occasional low temperature events,the seed-setting rate of tms5 increased to13%?15%,but the rate of k18 could be remained at 0.2%?1.9%,indicating that k18 has better performance of sterile stability under extreme weather conditions,which could increase the potential for agricultural production and application.Potassium Iodide staining showed ruptured pollen grains in k18 under high temperature and mature pollen under low temperature.SEM showed that the anthers in k18 were shriveled at high temperature,and most of pollen was aborted.Semi-thin sections indicated that the abnormal tapetum in k18 might be the cause of pollen abortion at stage 10 of anther development at high temperature.Further TEM result showed that the structures of Anther Cuticle,Ubisch body and Pollen Exine in k18 were similar to those of the wild-type(WT)at high temperature.However,from stage 10,the tapetum was less condensed cytoplasm and abnormal enlargement in k18,which finally resulted in pollen abortion.BF(Basic Fuchsin)and FB28(Fluorescent Brightener 28)staining showed that the Pollen Intine was absent at high temperature and recovered at lower temperature in k18.Given the tapetum in k18 is abnormal in later development stage of anther,we used TUNEL assay to analyze the Programmed Cell Death(PCD)process in WT and mutants.We can detect the fluorescent signal at stages8 and 9 of the WT,indicating that the tapetum PCD started in the tetrad period.In contrast,the k18 tapetum had no fluorescent signal in the above two periods,and the TUNEL signal was not detected until stage 11 at high temperature environment,indicating that the PCD initiation of k18 tapetum was delayed.q RT-PCR showed that the expressions of the tapetum specific transcriptional factor genes and sporopollenin synthesis genes in k18 were not significantly different from those of the WT.However,the expression of Os AP37,encoding an aspartic protease,was significantly different from WT,indicating that k18 affects tapetum PCD process in late developmental stage.The microspores in tapetal related mutants often exhibit degeneration at the mitosis stage.However,DAPI staining showed that k18 could still generate trinucleated pollen at high temperature,and all of these pollen grains were aborted eventually,which indicates that the tapetum still plays a crucial role at late stage of pollen development.This study provides a new genetic material with a novel thermo-sensitive sterile phenotype and facilitates a general understanding of plant reproduction. |
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