澳洲坚果花粉管荧光显微观察制片方法的对比分析  

Comparative analysis of slide preparation methods of Macadamia pollen tubes by fluorescence microscopic observation

作　　者：孔广红[1,2] 余文才 贺熙勇[1,2] 郑诚 吴超[1,2] 柳觐 KONG Guanghong;YU Wencai;HE Xiyong;ZHENG Cheng;WU Chao;LIU Jin(Yunnan Institute of Tropical Crops,Jinghong 666100,Yunnan,China;Yunnan Macadamia Agricultural Engineering Research Center,Jinghong 666100,Yunnan,China)

机构地区：[1]云南省热带作物科学研究所,云南景洪666100 [2]云南省澳洲坚果农业工程研究中心,云南景洪666100

出　　处：《中南林业科技大学学报》2023年第8期17-26,共10页Journal of Central South University of Forestry & Technology

基　　金：云南省热作所青年成长基金项目“澳洲坚果花粉管萌发特性的细胞和分子机制研究”(QNCZ2021-1);国家自然科学基金地区基金项目“澳洲坚果自交不亲和性S-RNase基因及其表达产物S糖蛋白的鉴定与作用机制研究”(31760215);云南省乡村振兴专项“云南省盈江县澳洲坚果产业科技特派团”(202104BI090004)。

摘　　要：【目的】探索荧光显微观察澳洲坚果花粉管原位生长的最佳制片方法,为澳洲坚果品种间杂交(不)亲和性及生殖生物学研究提供技术支撑。【方法】以品种‘HAES842’为母本、‘HAES863’为父本,异花授粉5 d后,采集雌蕊为试验材料,经过固定、软化、染色、镜检等步骤,比较分析不同制片方法下澳洲坚果花粉管的荧光显微观察效果。【结果】1)28℃下,NaOH溶液软化、徒手切片法中,选择1、2 mol·L^(-1)NaOH溶液对雌蕊软化,徒手切片容易获得雌蕊的完整组织;浓度为4、6 mol·L^(-1)时,雌蕊细胞反应剧烈,迅速失水后缓慢复水,切片时花柄易脱离,子房和柱头易破裂。2)100℃高温高压整体软化透明法中,NaOH溶液和Na2SO3溶液对澳洲坚果雌蕊均具有很好的软化效果,虽然镜检时花粉管被花柱其他组织遮挡,但此方法为整体观察澳洲坚果花粉管提出了新方向。3)未调节pH值和pH值7.0的染色液对澳洲坚果雌蕊进行染色,均未能明显区分花粉管和花柱其他组织;染色液pH值10.0时,花柱其他组织仅发微弱荧光,花粉管发黄绿色荧光,花粉管清晰明了,根根分明。【结论】28℃下,2 mol·L^(-1)NaOH溶液软化待检材料3 h,徒手切片,然后使用强碱性染液染色,能够很好地激发花粉管的黄绿色荧光信号,是澳洲坚果花粉管荧光显微观察的最佳制片方法,此方法能快速、清晰地观察澳洲坚果花粉管在花柱内的生长行为。【Objective】This study aimed to explore the best method of slide preparation for observing the in situ growth of pollen tubes by fluorescence microscopy,which can provide technical support for the study of compatibility or incompatibility and reproductive biology of Macadamia.【Method】This study used the Macadamia variety‘HAES842’as the female parent and‘HAES863’as the male parent,and the pistils at 5 days after cross-pollination were collected as the test material.After fixing,softening,dyeing,microscopy examination and further steps,the fluorescence microscopic observation effects of Macadamia pollen tubes by different methods of slide preparation were compared and analyzed.【Result】The results showed that:1)At 28℃,different concentrations of NaOH solutions were used to soften pistils,and it was easy to obtain the complete tissue of pistils by freehand sectioning when the concentration of NaOH solutions was 1 and 2 mol·L^(-1).The cells of pistils reacted violently,dehydrated rapidly and then rehydrated slowly when the concentration of NaOH solutions was 4 and 6 mol·L^(-1).At the same time,the flower stalks may be detached,and the ovaries and stigmas might be ruptured slightly after freehand sectioning of pistils.2)Both NaOH and Na2SO3 solutions had a good softening effect on the pistil of Macadamia at 100℃under high pressure.Although the pollen tube was blocked partly by additional tissues of the style during the microscopic examination,this method proposed a new sight for the whole pistil observation of the pollen tube of Macadamia.3)Pistils of Macadamia were dyed by pH-unadjusted and pH 7.0 water-soluble aniline blue solutions,but no pollen tubes from other tissues of style could be clearly distinguished.When the pH of the water-soluble aniline blue solution was 10.0,the internal tissue of the style only emitted weak fluorescence,the pollen tube emitted yellow-green fluorescence,and each pollen tube was extremely clear-1 and obvious.【Conclusion】In summary,the pistils of Macadamia we

关 键 词：澳洲坚果 花粉管 制片方法 软化 染色 

分 类 号：S718.46[农业科学—林学]