| In order to set up a protocol which contributed to observe wheat microspore development process and detect pollen fertility simply,precisely and quickly,under the conditions that wheat thermo-sensitive male sterile line BNS366 and its near-isogenic line Zhengmai 366 which was a common wheat cultivar were used as materials in this study.The microspore development process was observed and the pollen fertility was detected by optical microscopy.The self-seeds rate（national and international）was determined.The difference in the microspore development and the pollen fertility observation effect was analyzed among different treatments so as to get microscope images with increased recognization,firstly with 4 staining methods which included I2-KI method,acetate carmine method,haematoxylin method and carbolfuchsin method,secondly with 2 fixatives which included FAA and carnoys as I2-KI method,aceate carmine method and DAPI method were used for staining respectively,thirdly with 2 kinds of sampling methods which included taking anthers from upper,middle and lower positions spikelets or only from the middle spikelets as the pollens were stained with I2-KI method,aceate carmine method and DAPI method,respectively,and fourthly with 3 ethanol replacement treatments which included 90%-80%-70%,70%-70%and non-ethanol replacement after the wheat spikes fixed with FAA fixative as the pollens were stained with I2-KI method and DAPI method.On those foundation,the difference in the automatic counting application effect for the pollens was analyzed among 3 image analysis softwares which included Image J,Image-Pro Plus and Photoshop,so as to select the optimal software to provide a high throughput image analysis method for pollen fertility statistical work.The main results of this study were as follows:1.The BNS366 microspore abortion period was from the uninucleate period to the binucleate period.By the I2-KI method,the accumulation of starch in the microspore could be clearly reflected,but nucleus in microspore of Zhengmai 366 couldn’t be detected in trinucleate period and flowering period.The pollen fertility rate of Zhengmai 366 was noticeably higher than the self-seeds rate（national）in flowering period,and the operation was the easiest.By the acetate carmine method,the pollen fertility rate of Zhengmai 366was consistent with the self-seeds rate,nucleus could be showed clearly and and the operation was simple,as well as the accumulation of contents in the microspores could be seen but it was not clear.By the haematoxylin method,the staining effects were the best among the 4 methods in uninucleate period and binucleate period,but the operation was more complicated than I2-KI and acetate carmine method.With the carbol fuchsin method,the nucleus and contents could not be reflected clearly.Therefore,I2-KI method was the best for the accumulation of starch observation in the pollens,moreover cetate carmine method was the best for the changes observation of the nucleus and pollen fertility detection.2.The microspores fixed with FAA fixative,the starch accumulation area in microspore was stained deeper stained by I2-KI,and the nucleus were clearer stained by acetate carmine in uninucleate and binucleate period and by DAPI.After fixed with carnoys fixative,the nucleus were clearer stained by acetate carmine in trinucleate and flowering period,furthermore,the images were reddish in uninucleate and trinucleate period when the wheat microspores were stained by DAPI.In flowering period,as for Zhengmai 366,compared with the fertility rates of the microscopes stained by acetate carmine after fixed with FAA fixative and the self-seeds rate（national）,the fertility rates stained by I2-KI and DAPI with the two kinds of fixatives and by acetate carmine after fixed with carnoys fixatives were higher.Therefore,to obtain high-definition cell images of microscopes,FAA fixative was advisable for I2-KI method,and for acetate carmine method in uninucleate and binucleate period,and for DAPI method;as for carnoys fixative,it was advisable for acetate carmine method in trinucleate and flowering period.3.The pollen fertility rates with I2-KI method had no significant difference among the 2 sampling methods and the self-seeds rates（international）,and those were higher than the self-seeds rates（national）,except that there was no difference among the 4 indexes for BNS366 by early sowing,the same as below.The pollen fertility rates with acetic acid magenta method had no significant difference between the 2sampling methods.The pollen fertility rates of Zhengmai 366 by early sowing were in line with the self-seeds rates（national）and those were lower than the self-seeds rates（international）,however the pollen fertility rates of Zhengmai 366 by late sowing were in line with the self-seeds rates（international）and those were higher than the self-seeds rates（national）.The pollen fertility rates with DAPI method had no significant difference between the 2 sampling methods,and those were in line with commonly the self-seeds rates（international）and those were higher than the self-seeds rates（national）,especially when the pollens were from the middle spikelets anthers.Therefore,from a comprehensive point of view,from the perspective of saving the reagents required for sample fixation,replacement and storage,labor,reducing labor complexity,and accuracy of fertility in actual cytological observation,it was more appropriate to take the pollen in the middle spikelet for subsequent experiments,which could be used to self-seeds rate（internation）.From a scientific and rigorous point of view,researchers can also take the upper and lower pollen to tese the pollen fertility.4.The accumulation of starch in microspore could be clearly observed by I2-KI method under the three ethanol replacement strategies,and the order of the color depth of the starch accumulation area was as follows:non-replacement>70%-70%ethanol replacement>90%-80%-70%ethanol replacement.Under the treatment of 90%-80%-70%ethanol replacement,the nucleus status at different microspore development stages could be clearly shown by DAPI method,and cytoplasmic reddish was observed in BNS366 during the binucleate and trinucleate period,but that could not affect the cytological observation;Under the treatment of 70%-70%ethanol replacement,the nucleus were clearly shown by DAPI method,and cytoplasmic reddish was also observed in the trinucleate period of Zhengmai 366 and the binucleate period of BNS366,which were slightly worse than the treatment of 90%-80%-70%ethanol replacement,but that still did not affect the cytological observation;under the treatment of non-replacement,the images of Zhengmai 366 and BNS366 uninucleate and flowering period cells were clear,but the cytoplasm of Zhengmai 366 was stained in white and unclear,or in red and bright,even though some nucleus were stained in red and unclear,and the cytoplasm of BNS366 in micronuclei and trinuclear stage was red and intensified compared with the treatment of 70%-70%ethanol replacement.the order of cell image clarity was as follows:90%-80%-70%ethanol replacement>70%-70%ethanol replacement>non-replacement.Pollen fertility rates by I2-KI and DAPI method were consistent with the self-seeds rate（national）.To ensure the wheat microspore cytology observation,and to make the process cheaper,easier and safer than before,it was advisable for 70%-70%ethanol replacement strategy after FAA fixation.5.The rapid counting of pollen cells using 3 types of software was significantly more efficient than the manual counting method,but it was difficult to make the cell segmentation completely and clearly.There were a few inaccuracies.Among them,Image J had the best processing effect and the simplest operation,which could have batch processing and writing macro code for pollen fertility statistics,and the single image processing results were accurate,but batch processing had the problem of inaccurate automatic cell segmentation.Image-Pro Plus was the second in processing effect,although it could also implement various functions like Image J,but its writing macro operations was more complicated.Photoshop’s processing effect had no obvious advantages,because it had fewer functions in counting and only one more auxiliary counting function than manual counting,and it could not have batch processing like images.Therefore,whether it was for single or batch pictures in cell counting,Image J was the best choice,followed by Image-Pro Plus.Photoshop was not recommended in cell counting. |
