Endoreduplicative standards for calibration of flow cytometric C‐Value measurements

Abstract

It has been estimated that there are, globally, as many as 400,000 species of the angiosperms (the flowering plants). Of these, a minimal proportion has been characterized at the cytological level. Urgency is required in initiating a systematic and comprehensive census, due to species extinction as a consequence of anthropogenic activities. Fundamental to eukaryotes is the 2C-value, the amount of DNA contained within the nucleus of the unreduced gametes. Flow cytometry provides an ideal method for determining C-values, but the values archived in the Kew Plant C-value Database represent <2% of these species. Complicating the issue is a proliferation of different, and inconsistent standards for C-value measurements utilizing flow cytometry, and variability associated with different instrument platforms and using different staining procedures. In previous work, the use of flow cytometry for analysis of plant nuclear DNA contents for species spanning much of the range of genome sizes found in the angiosperms was described. For this work, an endoreduplicative species (Arabidopsis thaliana L.) was particularly helpful as an internal standard for genome size calibration. Such a standard is compromised if it overlaps in DNA content than that of the species whose genome size is sought. This report describes the use of a second species displaying endoreduplication, Capsicum annuum L., for similar standardization. The results (a) indicate accurate reporting of nuclear DNA contents across a range 0.32-423.68 pg, (b) confirm that endoreduplication increases nuclear DNA contents by complete replication of the genome, and (c) provide a means for quality control of linearity in instrumentation over defined dynamic ranges.