Novel whole-mount FISH analysis for intact root of Arabidopsis thaliana with spatial reference to 3D visualization

Whole-mount fluorescent in situ hybridization (WM-FISH) is an effective tool to observe chromosome behavior in tissues or organs. However, it is difficult to obtain a precise spatial profile of fluorescent signals in roots using conventional WM-FISH mainly because of the severe damage caused during the processing. To address this problem, we established a novel WM-FISH analysis for intact roots of Arabidopsis thaliana and successfully obtained a precise spatial profile of nuclear size and centromere signals. The two main improvements in the novel WM-FISH analysis are: (i) hybridization was performed directly on MAS-coated glass slides covered with silicon wells and (ii) conditions for enzyme treatment were optimized (37 degrees C, 45 s). After the WM-FISH using a centromere probe, we analyzed the results by 3D data processing to quantify the nuclear volume and number of centromere signals of the obtained cortical cell files and determined the position of each nucleus in intact roots. Then we plotted the nuclear volume and number of centromere signals versus distance from the quiescent center to evaluate the precise spatial profile of each parameter.

Automated summary

Whole-mount fluorescent in situ hybridization (WM-FISH) is a useful technique for studying chromosome behavior in tissues or organs. However, it is challenging to obtain precise spatial information in roots due to the damage caused during processing. In this study, a new approach was developed to perform WM-FISH on intact roots of Arabidopsis thaliana, allowing for accurate analysis of nuclear size and centromere signals. The method involved direct hybridization on MAS-coated glass slides and optimized enzyme treatment conditions. 3D data processing was utilized to quantify parameters and determine spatial profiles.