Kinematic Analysis using a Novel Method of Marking Roots with the Fluorescent Dye Calcofluor

in HortScience

Kinematic analysis allows accurate description of physiological changes along root axes by additionally taking into account changes due to dilution as cells expand. In previous studies using kinematic analysis, roots have been marked with ink by fine-tipped pens or single hair brushes. These methods have occasionally reduced root growth and limited resolution to the width of the marks, usually 1 mm. We describe a new method of marking roots with the fluorescent dye calcofluor which does not reduce root growth. The terminal 7 mm of bean root tips were grown vertically in a glass chamber into which a constant flow of aerated nutrient solution was passed. A 0.001% calcofluor solution was pulsed through the chamber for 1 min. Excess calcofluor was removed rapidly by a high rate of nutrient flow (200 ml·h–1) for 3 min. after which flow was reduced to 20 ml/hr. Roots were magnified 11.5× under a microscope mounted horizontally and five digitized images captured every 5 min. Imaging software allowed determination of fluorescence of individual pixels along the length of the root. Fluorescence decreased in the zone of cell elongation due to dilution as cells expanded. This method may improve resolution of kinematic analysis to the length of individual pixels, which was 18 microns at 11.5× magnification.

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