S. Bauwens et al., PROCEDURE FOR WHOLE-MOUNT FLUORESCENCE IN-SITU HYBRIDIZATION OF INTERPHASE NUCLEI ON ARABIDOPSIS-THALIANA, Plant journal, 6(1), 1994, pp. 123-131
A procedure for whole mount fluorescence in situ hybridization (FISH)
on plant tissue is reported. The technique was demonstrated on seedlin
gs and flowers of Arabidopsis thaliana L. with rDNA as a probe, labell
ed, both for direct and indirect detection. It was found that fixation
in 1% formaldehyde yielded the best results with respect to morpholog
y and hybridization efficiency. The combination of whole mount FISH an
d confocal scanning laser microscopy allowed the nuclear localization
of the rDNA loci in all tissues of both seedlings and flowers. Direct
labelling yielded the best signal-to-noise ratio, especially in the ap
ical zones of the seedlings. The technique was further illustrated on
seedlings of A, thaliana in double labelling experiments with rDNA and
a tandemly repeated, 500 bp sequence of A. thaliana. Although nuclei
in all tissues in the seedling exhibited both signals, hybridization e
fficiency for both signals was reduced in the dense, apical zones as c
ompared with single labelling experiments with rDNA.