陆地棉脱外壁花粉粒的制备和花粉粒核DNA检测

Preparation of De-exined Pollen Grains and Observation of Pollen Grains Nuclear DNA in Gossypium hirsutum L.

以陆地棉品种苏棉22号的花粉为材料,研究棉花花粉粒脱外壁和检测花粉粒内部核DNA状态的方法。花粉粒经4%多聚甲醛固定后,通过10%次氯酸钠水溶液氧化、55℃热激30min、压片等系列程序可将花粉粒的外壁完全脱去,分离出完整的脱外壁花粉粒。用DNA荧光染料DAPI分别对陆地棉花粉粒和脱外壁花粉粒进行染色,在荧光显微镜和激光扫描共聚焦显微镜下观察花粉粒的内部核结构。没有脱外壁的花粉粒的内部核结构难以被观察到,而脱外壁花粉粒的核DNA结构清晰可见,内壁几乎无荧光。首次发现陆地棉的成熟花粉粒为三核花粉粒。

The objective of this study was demonstrating nuclei DNA in the pollen grains of upland cotton （Gossypium hirsutum cυ. Sumian 22 ）. The procedures of detaching the exine and observing nuclei DNA of pollen grains were established. The pollen grains were fixed with 4% paraformaldehyde solution, oxidated with 10% sodium hypochlorite solution, heat shocked at 55℃ for 30 minutes,then the slide with pollens solution was pressed by hand. Finally, the exine of pollen grains could be detached. The pollen grains and de-exined pollen grains were stained with DAPI and observed with fluo-rescence microscope and laser scanning confocal microscope （Leica TCS SP2）, respectively. The nucleus of cotton pollen grain could not be observed, but the nuclear DNA of de-exined pollen grain could be observed clearly. The inner wall fluorescence of pollen grain was not found. The three nuclei of the pollen grain of upland cotton cultivar Sumian 22 were observed in the experiment.