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

Abstract

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).