Sensitivity enhancement of fluorescence in situ hybridization on plant chromosomes

An improved in situ hybridization procedure is presented, based on synchronization of root meristems of barley and wheat, enzymatic digestion, a protoplast drop technique, and the use of the fluorescent dye Cy3. The combination of these approaches resulted in a significant increase of well-spread metaphases suitable for in situ hybridization as compared to squash preparations, and to a significantly enhanced number and intensity of hybridization signals as demonstrated for a B-hordein-specific low-copy probe of barley. In the case of Cy3 all metaphases displayed a signal, more than 60% of them on both chromatids of each gene-bearing chromosome.