A modified method for preparing meiotic chromosomes based on digesting pollen mother cells in suspension

Abstract

BackgroundMeiotic chromosome preparation is a key step in plant meiotic research. Pollen mother cell (PMC) wall elimination is beneficial to cytogenetic experimental procedures. Without wall interference, these procedures are easier and more successful. In existing methods it is difficult to eliminate PMC walls completely and uniformly. In this paper, we present an improved method for digesting PMC walls, and one for providing massive chromosomal spreads on a slide for other cytogenetic experimental procedures.ResultsThree plants were selected to exhibit the modified meiotic chromosome preparation method. PMCs were dispersed as single cells and incubated in a mixed enzyme solution (3 % cellulose + 0.3 % pectinase + 1 % snailase) for 1.5–2.5 h. In total, 28.28 % cells were lost during this process. There were 800–1900 spreads on every slide and no PMC wall interference was found on any of the slides. The spreads were also evenly distributed on the slides. More spreads were obtained when PMC and protoplast densities in the suspension were increased. All three plants’ spreads were successfully used to locate a 5 s rDNA conserved sequence. The Nicotiana hybrid’s spreads were successfully used to identify the hybrid’s parental genome.ConclusionThis is an alternative method for meiotic chromosome preparation. Through this method, PMC walls can be completely and uniformly eliminated, and hundreds of spreads on every slide can be obtained. These spreads can be successfully used for DNA in situ hybridization.

Application of a modified drop method for high-resolution pachytene chromosome spreads in two Phalaenopsis species

“…The drop method was first developed for preparing chromosome spreads of human cells [ 29 ] and for protoplast isolation in plant species [ 30 ]. Since then, several methods have been published to improve the techniques of meiotic chromosome preparation for plant species [ 19 , 28 , 31 , 32 ], but none of these methods has been reported suitable for orchid species with specialized pollinia. In this study, we developed the first easily mastered drop method for preparation of high-quality meiotic pachytene chromosomes in Phalaenopsis species.…”

Research Article Contributions of classical and molecular cytogenetic in meiotic analysis and pollen viability for plant breeding.

“…The FISH extends the power of resolution to the chromatin display, providing essential information for the study of chromosome behavior in meiosis, data that cannot be obtained by classical cytogenetic application techniques (Dang et al, 2015). In the meiotic analysis, the observation of numerical chromosomal changes such as nullisomics (Ji et al, 1997) and genome structure (Scholes, 2011) has been facilitated by the identification of homologous chromosomes using rDNA probes, genomic regions commonly found and used in the comparative meiotic analysis.…”