Pan Gang, Zhou Yongming, Fowke Larry C, Wang Hong
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China.
Plant Cell Rep. 2004 Oct;23(4):196-202. doi: 10.1007/s00299-004-0830-y. Epub 2004 Jul 28.
A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.
开发了一种简单可靠的方法，用于从甘蓝型油菜和普通小麦中分离花粉核，以便使用流式细胞术进行DNA分析。通过超声处理从花粉中释放细胞核。经过尼龙网过滤和纯化程序后分离出的细胞核适用于流式细胞术分析以及基因组DNA的分离。针对甘蓝型油菜不同发育阶段的花粉优化了超声处理时间。该方法有效且适用于多个样品的制备。我们分析了甘蓝型油菜三个主要发育阶段花粉以及成熟小麦花粉中的核DNA水平。从甘蓝型油菜单核小孢子分离出的细胞核以及成熟小麦花粉中仅检测到代表单倍体DNA水平的单个1C峰。有趣的是，在甘蓝型油菜的双核花粉和成熟花粉中均检测到二倍体细胞核。讨论了二倍体细胞核的可能来源。
