不同固定液对小麦小孢子发育和花粉育性检测的影响
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摘要
为了明确FAA和卡诺氏固定液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I_2-KI法、醋酸洋红法和DAPI法染色观察了小孢子发育过程,并统计了花粉可育率和自交结实率。结果表明,采用FAA固定液固定、I_2-KI法染色,小孢子内淀粉积累区域染色较深,单核期和二核期醋酸洋红法与DAPI法染色细胞核清晰程度均较高;采用卡诺氏固定液固定、三核期和开花期醋酸洋红法染色,细胞核清晰度较高,二核期与三核期DAPI法细胞图像略偏红。采用两种固定液固定、I_2-KI法和DAPI法染色,以及采用卡诺氏固定液固定、醋酸洋红法染色,花粉可育率均较高;采用FAA固定液固定、醋酸洋红法染色,花粉可育率偏低并与自交结实率一致。因此,从获得小孢子高清晰细胞图像角度考虑,对于I_2-KI、单核期和二核期醋酸洋红及DAPI染色法来说,采用FAA固定液较好;对于三核期和开花期醋酸洋红染色法来说,采用卡诺氏固定液较好。
In order to clarify the differences in the staining effects of the microspores after the fixation of FAA and carnoys fixatives,the thermo-sensitive male-sterile wheat line BNS 366 and its near-isogenic line Zhengmai366 were used as the materials,and the microspore development process was investigated by I_2-KI method,acetate carmine method and DAPI method. Moreover,the pollen fertility at flowering stage and the selfing seedset rate were measured. The results showed that for the microspores fixed with FAA fixative,the starch accumulation area in microspore was heavily stained by I_2-KI,and the nuclei were lightly stained by acetate carmine and DAPI in uninucleate and binucleate periods. When fixed with carnoys fixative,the nuclei were lightly stained by acetate carmine in trinucleate and flowering periods,and the images were reddish in uninucleate and trinucleate periods when the wheat microspores were stained by DAPI. Pollen fertility was higher when two kinds of fixatives combined with I_2-KI or DAPI staining and acetate carmine staining after fixed with carnoys fixatives were used,Pollen fertility was low and the self-fertility rate was consistent with that of self-pollination by FAA fixation and acetate carmine staining. Therefore,to obtain high-definition cell images of microscopes,FAA fixative is advisable for I_2-KI method,and for acetate carmine method in uninucleate and binucleate period,and for DAPI method; as for carnoys fixative,it is advisable for acetate carmine method in trinucleate and flowering period.
In order to clarify the differences in the staining effects of the microspores after the fixation of FAA and carnoys fixatives,the thermo-sensitive male-sterile wheat line BNS 366 and its near-isogenic line Zhengmai366 were used as the materials,and the microspore development process was investigated by I_2-KI method,acetate carmine method and DAPI method. Moreover,the pollen fertility at flowering stage and the selfing seedset rate were measured. The results showed that for the microspores fixed with FAA fixative,the starch accumulation area in microspore was heavily stained by I_2-KI,and the nuclei were lightly stained by acetate carmine and DAPI in uninucleate and binucleate periods. When fixed with carnoys fixative,the nuclei were lightly stained by acetate carmine in trinucleate and flowering periods,and the images were reddish in uninucleate and trinucleate periods when the wheat microspores were stained by DAPI. Pollen fertility was higher when two kinds of fixatives combined with I_2-KI or DAPI staining and acetate carmine staining after fixed with carnoys fixatives were used,Pollen fertility was low and the self-fertility rate was consistent with that of self-pollination by FAA fixation and acetate carmine staining. Therefore,to obtain high-definition cell images of microscopes,FAA fixative is advisable for I_2-KI method,and for acetate carmine method in uninucleate and binucleate period,and for DAPI method; as for carnoys fixative,it is advisable for acetate carmine method in trinucleate and flowering period.
引文
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[11]赵卜,张向展,郑炜君,等.F型小麦雄性不育系小孢子发育的细胞学观察[J].麦类作物学报,2015,35(7):919.ZHAO B,ZHANG X Z,ZHENG W J,et al.Cytological observation on meiosis and development of microspores in F-type male sterile wheat line [J].Journal of Triticeae Crops,2015,35(7):919.
[12]王洪燕.利用DNA荧光探针DAPI检测番茄花粉粒的发育时期[J].枣庄学院学报,2006,23(2):94.WANG H Y.Detection of the developmental stage of tomato pollen grains by DNA fluorescent probe DAPI [J].Journal of Zaozhuang University,2006,23(2):94.
[13]王小利,张改生,陈新宏,等.两类二角山羊草细胞质小麦雄性不育系的细胞学研究[J].西北植物学报,2004,24(5):782.WANG X L,ZHANG G S,CHEN X H,et al.Study on cytology of two male sterile wheats with Aegilops bicornis cytoplasm [J].Acta Botanica Boreali-occidentalia Sinica,2004,24(5):782.
[14]钱焕焕,罗钊,李艳丽,等.YS型小麦温敏不育系A731雄性败育的细胞学研究[J].麦类作物学报,2012,32(6):1027.QIAN H H,LUO Z,LI Y L,et al.Cytological studies on male fertility abortion of YS type thermo-sensitive male sterile wheat line A731 [J].Journal of Triticeae Crops,2012,32(6):1027.
[2]谭昌华,余国东,杨沛丰,等.重庆温光型核不育小麦的不育性研究初报[J].西南农业学报,1992,5(4):1.TAN C H,YU G D,YANG P F,et al.Preliminary study on sterility of thermo-photo-sensitive genic male sterile wheat in Chongqing [J].Southwest China Journal of Agricultural Sciences,1992,5(4):1.
[3]赵昌平,张立平,李云伏,等.小麦光温敏雄性不育相关基因的DDRT-PCR分析及功能检测[J].中国生物化学与分子生物学报,2007,23(1):57.ZHAO C P,ZHANG L P,LI Y F,et al.Analysis of photoperiod-temperature sensitive male sterility related genes in wheat with DDRT-PCR [J].Chinese Journal of Biochemistry and Molecular Biology,2007,23(1):57.
[4]杨木军,顾坚,刘琨,等.小麦温光敏雄性核不育系K78S在云南的生态适应性研究[J].作物学报,2006,32(11):1619.YANG M J,GU J,LIU K,et al.Ecological adaptability of thermos-photo-sensitive genic male sterile wheat K78S in Yunan Province [J].Acta Agronomica Sinica,2006,32(11):1619.
[5]张建奎,董静,宗学凤,等.温光敏核雄性不育小麦雄性不育的细胞学观察[J].植物遗传资源学报,2009,10(3):411.ZHAGN J K,DONG J,ZONG X F,et al.Cytological analysis of pollen abortion in thermos/photoperiod-sensitive genic male sterile wheat(Triticum aestivum L) [J].Journal of Plant Genetic Resources,2009,10(3):411.
[6]李式昭,曹墨菊,荣廷昭,等.太空诱变玉米核不育材料花粉败育的细胞学观察[J].分子细胞生物学报,2007,40(5):359.LI S Z,CAO M J,RONG Y Z,et al.Cytological observation on pollen abortion of genetic male sterile mutant induced by space flight in maize [J].Journal of Molecular Cell Biology,2007,40(5):359.
[7]贺晓敏,周美兰,余传启,等.温敏雄性不育小麦BNS366花粉败育的细胞学观察[J].麦类作物学报,2014,34(4):461.HE X M,ZHOU M L,YU C Q,et al.Cytological observation on pollen male fertility abortion of thermo sensitive male sterile wheat line BNS366 [J].Journal of Triticeae Crops,2014,34(4):461.
[8]刘子涵,蒙立颖,姚盟,等.牡山羊草细胞质雄性不育小麦败育的生物学特性和细胞学研究[J].中国农业大学学报,2016,21(5):5.LIU Z H,MENG L Y,YAO M,et al.Biological characteristics and cytological studies on abortion of Aegilops juvenalis cytoplasmic male sterile line [J].Journal of China Agricultural University,2016,21(5):5.
[9]黄兴国,汪广勇,余金洪,等.水稻同核异质雄性不育系的细胞质遗传效应与细胞学研究[J].中国水稻科学,2011,25(4):376.HUANG X G,WANG G Y,YU J H,et al.Cytoplasmic genetic effects and cytological study of isonuclear alloplasmic male sterile lines in rice [J].China Journal of Rice Science,2011,25(4):376.
[10]刘海英,甄俊琦,胡铁柱,等.小麦温敏雄性不育系BNS366小孢子发育和花粉育性检测方法研究[J].麦类作物学报,2018,38(4):380.LIU H Y,ZHEN J Q,HU T Z,et al.Study on microspore development and pollen fertility detection of thermo-sensitive male-sterile wheat line BNS366 [J].Journal of Triticeae Crops,2018,38(4):380.
[11]赵卜,张向展,郑炜君,等.F型小麦雄性不育系小孢子发育的细胞学观察[J].麦类作物学报,2015,35(7):919.ZHAO B,ZHANG X Z,ZHENG W J,et al.Cytological observation on meiosis and development of microspores in F-type male sterile wheat line [J].Journal of Triticeae Crops,2015,35(7):919.
[12]王洪燕.利用DNA荧光探针DAPI检测番茄花粉粒的发育时期[J].枣庄学院学报,2006,23(2):94.WANG H Y.Detection of the developmental stage of tomato pollen grains by DNA fluorescent probe DAPI [J].Journal of Zaozhuang University,2006,23(2):94.
[13]王小利,张改生,陈新宏,等.两类二角山羊草细胞质小麦雄性不育系的细胞学研究[J].西北植物学报,2004,24(5):782.WANG X L,ZHANG G S,CHEN X H,et al.Study on cytology of two male sterile wheats with Aegilops bicornis cytoplasm [J].Acta Botanica Boreali-occidentalia Sinica,2004,24(5):782.
[14]钱焕焕,罗钊,李艳丽,等.YS型小麦温敏不育系A731雄性败育的细胞学研究[J].麦类作物学报,2012,32(6):1027.QIAN H H,LUO Z,LI Y L,et al.Cytological studies on male fertility abortion of YS type thermo-sensitive male sterile wheat line A731 [J].Journal of Triticeae Crops,2012,32(6):1027.