Enzymatic Treatment of Plant Material to Spread Chromosomes for In Situ Hybridization

An important factor contributing to the success of
DNA
:DNA
in situ
hybridization is the preparation of clean chromosome spreads (
1
,
2
). The chromosomes and nuclei should be well separated and free of cytoplasm, debris, and dirt (Fig. 1 ). Nonspecific signal is deposited on cytoplasm and cell debris, and cytoplasm can mask the chromosomes and hinder the access of probe and detection reagents. This chapter describes two techniques for preparing chromosome spreads. The technique for preparing chromosomes by the squashing method (Section 3.2. ) is modified from Schwarzacher et al. (
3
), and the dropping method (Section 3.3. ) from Ambros et al. (
1
) and Geber and Schweizer (
4
).Fig. 1.
Phase contrast micrograph of a squashed root tip meristem after enzyme digestion. Most of the cell walls have been degraded and the nuclei and chromosomes (dark gray to black) are liberated. Little cytoplasm and debris (lighter gray) are visible. Bar = 10 �m.