Isolation of plant nuclei suitable for flow cytometry from recalcitrant tissue by use of a filtration column |
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Authors: | Hsiao-Ching Lee Tsai-Yun Lin |
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Institution: | (1) Department of Life Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, 30013 Hsinchu, Taiwan |
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Abstract: | Flow cytometry is widely applied in the determination of nuclear DNA content and ploidy level in many organisms. However,
a difficulty with flow cytometry is the method's intrinsic inability to tolerate large particles that associate with the isolated
nuclei. A suspension of plant nuclei can often contain a high level of crystalline calcium oxalate, which blocks the fluidics
system of the flow cytometer. We designed a cotton column and added polyvinylpyrrolidone-40 to the buffer to remove phenolic
impurities and cytoplasmic compounds from plant nuclei, making the suspension suitable for flow cytometry. This simple and
highly efficient protocol enables isolation of intact nuclei from plant tissues containing high levels of polysaccharides,
calcium oxalate crystals and other metabolites. Our protocol resulted in the isolation of intact nuclei from mature orchid
leaves. This method can be used on recalcitrant tissues and is particularly effective on plants containing calcium oxalate
crystals. |
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Keywords: | endoreduplication flow cytometry nuclei isolation Phalaenopsis polyploidy |
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