Ploidy determination using flow cytometry

Abstract

Measuring ploidy level is of highest importance in the final stages of haploid induction programs. For breeding purposes, a large number of haploids usually need to be tested, so an efficient ploidy analysis is a prerequisite for successful application. Methods to determine ploidy level may be direct (chromosome counting) or indirect (flow cytometry, stomatal size, chloroplast number of the guard cells and morphological observations). Various opinions about the usefulness of the mentioned techniques can be found. Sari et al. (1999) for instance, comparing various ploidy measurement methods in haploid watermelons concluded that “counting chromosomes is cumbersome, producing plants for morphological observations requires a long time and flow cytometry is expensive and labour intensive.” They proposed that “measurement of stomata and chloroplast counting methods are simple to use and less labor intensive, and hence can be considered a practical alternative to the others.” The author’s experiences are based on flow cytometric measurements of a large number of haploid regenerants obtained mainly by androgenesis (cabbage), gynogenesis (onion) or distant fertilization (potato), as well as on genome size analysis of various species. Several points will be made to explain why, in the author’s opinion, for ploidy analysis of haploid regenerants, flow cytometry is of much higher relevance than any other proposed method. The main aim of this article is to explain the basic features of flow cytometry and to propose optimized protocols for rapid and simple flow cytometric evaluations using either a HBO lamp or laser equipped flow cytometers.