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

## TL;DR

This paper introduces an improved method for preparing meiotic chromosomes in plants by effectively removing pollen mother cell walls, enabling better cytogenetic studies.

## Contribution

The novel contribution is a modified method for uniform and complete PMC wall digestion, enabling high-quality chromosomal spreads for downstream experiments.

## Key findings

- The method produced 800–1900 chromosomal spreads per slide with no PMC wall interference.
- The method successfully located a 5s rDNA conserved sequence in three plant species.
- Increased PMC and protoplast density in suspension resulted in more chromosomal spreads.

## Abstract

Meiotic 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.

Three 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.

This 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.