Neutral red as a probe for confocal laser scanning microscopy studies of plant roots

Abstract

Background and Aims Neutral red (NR), a lipophilic phenazine dye, has been widely used in various biological systems as a vital stain for bright-field microscopy. In its unprotonated form it penetrates the plasma membrane and tonoplast of viable plant cells, then due to protonation it becomes trapped in acidic compartments. The possible applications of NR for confocal laser scanning microscopy (CLSM) studies were examined in various aspects of plant root biology. Methods NR. was used as a fluorochrome for living roots of Phaseolm vulgaris, Allium cepa, A. porrum and Arabidopsis thaliana (wild-type and transgenic GFP-carrying lines). The tissues were visualized using CLSM.. The effect of NR on. the integrity of the cytoskeleton and the growth rate of arabidopsis primary roots was analysed to judge potential toxic effects of the dye. Key Results The main, advantages of the use of NR are related to the fact that NR rapidly penetrates root tissues, has affinity to suberin and lignin, and accumulates in the vacuoles. It is shown that NR is a suitable probe for visualization of proto- and metaxylern. elements, Casparian. bands in the en.doder.mis, and vacuoles in cells of living roots. The actin cytoskeleton and the microtubule system of the cells, as well as the dynamics of root growth, remain unchanged after short-term application of NR, indicating a relatively low toxicity of this chemical. It was also found that NR. is a useful probe for the observation of the internal structures of root nodules and of fungal hyphae in vesicular-arbuscular mycorrhizas. Conclusions Ease, low cost and absence of tissue processing make NR a useful, probe for structural, developmental. and vacuole-biogenetic studies of plant roots with CLSM.