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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Abbreviations
- PVP:
-
polyvinylpyrrolidone
References
Arumuganathan K and Earle ED (1991) Estimation of nuclear DNA content of plants by flow cytometry. Plant Mol Biol Rep 9: 229–233.
De Rocher EJ, Harkins KR, Galbraith DW, and Bohnert HJ (1990) Developmentally regulated systemic endopolyploidy in succulents with small genomes. Science 250: 99–101.
Dolezel J and Bartos J (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95: 99–110.
Galbraith DW, Harkins KR, and Knapp S (1991) Systemic endopolyploidy inArabidopsis thaliana. Plant Physiol 96: 985–989.
Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, and Firoozabady E (1983) Rapid flow cytophotometric analysis of the cell cycle in intact plant tissues. Science 220: 1049–1051.
Gilissen LJW and van Staveren MJ and Creemers-Molenaar J, and Verhoeven HA (1993) Development of polysomaty in seedlings and plants ofCucumis sativus L. Plant Sci 91: 171–179.
Larkins BA, Dilkes BP, Dante RA, Coelho CM, Woo YM, and Liu Y (2001) Investigating the hows and whys of DNA endoreduplication, J Exp Bot 52: 183–192.
Lee HC, Chiou DW, Chen WH, Markhart AH, Chen YH, and Lin TY (2004) Dynamics of cell growth and endoreduplication during orchid flower development. Plant Sci 166: 659–667.
Lin S, Lee HC, Chen WH, Chen CC, Kao YY, Fu YM, Chen YH, and Lin TY (2001) Nuclear DNA contents ofPhalaenopsis sp. andDoritis pulcherrima. J Amer Soc Hort Sci 126: 195–199.
Schweizer L, Yerk-Davis GL, Phillips RL, Srienc F, and Jones RJ (1995) Dynamics of maize endosperm development and DNA endoreduplication. Proc Natl Acad Sci USA 92: 7070–7074.
Smulders MJM, Rus-Kortekaas W, and Gilissen LJW (1994) Development of polysomaty during differentiation in diploid and tetraploid tomato (Lycopersicon esculentum) plants. Plant Sci 97: 53–60.
Traas J, Hülskamp M, Gendreau E, and Höfte H (1998) Endoreduplication and development: rule without dividing? Curr Opin Plant Biol 1: 498–503.
Ulrich I and Ulrich W (1991) High-resolution flow cytometry of nuclear DNA in higher plants. Protoplasma 165: 212–215.
Yang M and Loh CS (2004) Systemic endopolyploidy inSpathoglottis plicata (Orchidaceae) development. BMC Cell Biol 5: 33.
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Lee, HC., Lin, TY. Isolation of plant nuclei suitable for flow cytometry from recalcitrant tissue by use of a filtration column. Plant Mol Biol Rep 23, 53–58 (2005). https://doi.org/10.1007/BF02772646
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DOI: https://doi.org/10.1007/BF02772646