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作者	单位
刘海英，甄俊琦，茹振钢，张亚娟，冯必得，景秀秀，王莹	（1.河南师范大学生命科学学院，河南新乡 453007；2.河南科技学院小麦中心，河南新乡 453003）

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中文摘要:

为了探究小麦穗经FAA固定液固定后，不同乙醇置换处理对小麦小孢子花粉染色效果的差异，以温敏雄性不育系BNS366及其近等基因系郑麦366为材料，采用90%-80%-70%、70%-70%和不置换3种乙醇置换方式处理后，用I₂-KI法和DAPI法分别染色对小孢子发育过程进行了观察，并统计了花粉可育率和自交结实率。结果表明，3种乙醇置换处理下，I₂-KI法染色均可以清晰显示小孢子内淀粉积累情况，其淀粉积累区域颜色由深到浅排序为不置换>70%-70%乙醇置换>90%-80%-70%乙醇置换。在90%-80%-70%乙醇置换处理下，DAPI法染色在小孢子不同发育时期均能清晰显示细胞核状况，在BNS366的二核期和三核期有细胞质微红现象，但不影响细胞学观察；在70%-70%乙醇置换处理下，DAPI法染色细胞核清晰，在郑麦366三核期和BNS366的二核期也存在细胞质微红现象且程度略有加重，仍不影响细胞学观察；在不置换处理下，DAPI法染色郑麦366和BNS366单核期和开花期的小孢子细胞图像清晰，郑麦366三核期小孢子细胞质发白浑浊，或发红发亮，甚至部分细胞核被染成红色，核显示不清晰，BNS366二核期和三核期小孢子细胞质着红色且比70%-70%乙醇置换处理下程度加剧。三种乙醇置换处理按细胞图像清晰度排序为90%-80%-70%乙醇置换>70%-70%乙醇置换>不置换。I₂-KI和DAPI染色统计的花粉可育率与其自交结实率基本一致。从小麦小孢子细胞学观察效果，兼顾经济实用和试验安全角度考虑，FAA固定后采用70%-70%乙醇置换处理较为可取。

英文摘要:

In order to investigate the differences in the staining effects of the micropores under three ethanol replacement treatments(90%-80%-70%,70%-70% and no replacement),after the fixation of formaldehyde-acetic acid-ethanol(FAA),the thermo-sensitive male-sterile wheat line BNS366 and its near-isogenic line Zhengmai 366 were used as the materials,and the microspore development process was investigated by I₂-KI method and DAPI method. The pollen fertility at flowering stage and the selfing seedsetting rate were measured. The results showed that the accumulation of starch in microspore could be clearly observed by I₂-KI method under the three ethanol replacement treatments,and the order of the color of the starch accumulation area ranked as non-replacement>70%-70% ethanol replacement> 90%-80%-70% ethanol replacement. Under the treatment of 90%-80%-70% ethanol replacement,the nuclear status at different microspore development stages could be clearly shown by DAPI method,and cytoplasmic reddiness was observed in BNS366 during the binucleate and trinucleate periods,but that would not affect the cytological observation. Under the treatment of 70%-70% ethanol replacement,the nuclei were clearly shown by DAPI method,and cytoplasmic reddiness was also observed in the trinucleate period of Zhengmai 366 and in the binucleate period of BNS366,slightly worse than that under 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 cells at uninucleate and flowering periods 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. The cytoplasm of BNS366 at micronuclei and trinuclear stages was red and intensified compared with that under the treatment of 70%-70% ethanol replacement. The order of three ethanol replacement treatments according to the clarity of cell image was:90%-80%-70% ethanol replacement > 70%-70% ethanol replacement>non-replacement. The results of I₂-KI and DAPI staining showed that the fertility of pollen was basically the same as that of self pollination. In view of the observation effect of wheat microspore cytology,economy,practicality and test safety,70%-70% ethanol replacement after FAA fixation was preferable.

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