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(American Journal of Botany. 1999;86:609-613.)
© 1999 Botanical Society of America, Inc.

Reference standards for determination of DNA content of plant nuclei1

J. Spencer Johnston 2 , Michael D. Bennett 3 , A. Lane Rayburn 4 , David W. Galbraith 5 and H. James Price 6, 7

Departments of 2Entomology and 6Soil and Crop Sciences, Texas A&M University, College Station, Texas 77843; 3Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK; 4Department of Agronomy, University of Illinois, Urbana, Illinois 61821; and 5Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721

Flow cytometry was used to compare 14 potential reference standards for plant DNA content determination. Both chicken and plant internal standards were used, as were propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) as fluorochromes. Means and standard errors of the means are presented for the 14 potential reference standards, and the means are compared to those obtained by Feulgen densitometry. Five species are recommended as an initial set of international standards for future plant DNA content determinations: Sorghum bicolor cv. Pioneer 8695 (2C = 1.74 pg), Pisum sativum cv. Minerva Maple (2C = 9.56 pg), Hordeum vulgare cv. Sultan (2C = 11.12 pg), Vicia faba (2C = 26.66 pg), and Allium cepa cv. Ailsa Craig (2C = 33.55 pg). It is recommended that the reference standard of choice be one with 2C and 4C nuclear DNA content peaks similar to, but not overlapping, the 2C and 4C peaks of the target species. We recommend PI as the fluorochrome of choice for flow cytometric determination of plant DNA content. DAPI should be used only if the estimated DNA value is corroborated by using a second stain that has no bias for AT- or GC-rich sequences within genomes.

Key Words: DAPI • DNA content • flow cytometry • propidium iodide • reference standards




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