An efficient procedure for isolation of nuclei from plant protoplasts
An efficient procedure for isolation of nuclei from plant protoplasts
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DOI：
10.1007/bf01276340
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发表时间：
1985-06
期刊：
影响因子：
2.9
通讯作者：
P. Saxena;L. Fowke;J. King
中科院分区：
文献类型：
--
作者：
P. Saxena;L. Fowke;J. King
本文介绍了一种简便、有效、快速的从植物原生质体中分离细胞核的方法。通过在含有极低浓度（0.01%）去污剂Triton X-100的适当缓冲液中破坏原生质体来完成细胞核的释放。细胞核分离缓冲液的pH值（5.3）在大量稳定细胞核的回收中起关键作用。用精胺（0.1 mM）、二硫苏糖醇（2.5 mM）、乙二胺四乙酸（2.5 mM）和NAD和KCl（各10 mM）补充缓冲液（10 mM MES）提高了核产率和质量。利用所开发的方法，可以在30分钟内从原生质体中常规回收95%的细胞核。核制备物具有高纯度，几乎没有可检测到的细胞质污染，并且没有核聚集。通过Nomarski微分干涉对比光学和超微结构观察，对核的结构完整性进行了评估和证实。
The present communication describes an easy, efficient and rapid method for isolation of nuclei from plant protoplasts. Release of nuclei is accomplished by disruption of protoplasts in an appropriate buffer containing a very low concentration (0.01%) of the detergent Triton X-100. The pH of the nuclei isolation buffer (5.3) played a critical role in the recovery of stable nuclei in large numbers. Supplementation of buffer (10 mM MES) with spermine (0.1 mM), dithiothreitol (2.5 mM), ethylenediaminetetraacetic acid (2.5 mM) and Nad and KCl (10 mM each) improved nuclear yield and quality. With the method developed it is possible to routinely recover 95% nuclei from the protoplasts within 30 minutes. The nuclear preparations are of high purity with little detectable cytoplasmic contamination and no clumping of the nuclei. The structural integrity of the nuclei has been assessed and confirmed by Nomarski differential interference contrast optics and ultrastructural observations.