Abstract
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.
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