In Vivo Ploidy Determination of Arabidopsis thaliana Male and Female Gametophytes.

Abstract

Organ- or tissue-specific ploidy level determination is often used for answering biological, molecular, genetic, or evolutionary questions in plant sciences. However, current techniques for ploidy determination either cannot provide information on single cell level, require destructive sample preparation, or are laborious and time-consuming. Here, we present a new approach developed in Arabidopsis thaliana, which is not only less labor intensive but also allows in vivo ploidy determination on single cell level. The technique is based on the incorporation of a transgenic construct, consisting of the centromere-specific protein CENH3 fused to the fluorescent reporter GFP that specifically labels centromeric regions and hence allows for an accurate visual determination of the cell's chromosome number. Moreover, by combining the construct with a gametophyte-specific promoter, the technique enables accurate chromosome quantification in all individual gametophytic cell types formed during micro- and mega-gametogenesis. As such, CENH3-based centromere visualization provides an easy and straightforward method to monitor meiotic cell division integrity, gametophytic chromosome dynamics, and reproductive ploidy stability.