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AOBPreview originally published online on March 6, 2006
Annals of Botany 2006 97(6):1127-1138; doi:10.1093/aob/mcl045
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© The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

TECHNICAL ARTICLE

Neutral Red as a Probe for Confocal Laser Scanning Microscopy Studies of Plant Roots

JOSEPH G. DUBROVSKY1,*, MARTIN GUTTENBERGER2, ANDRES SARALEGUI3, SELENE NAPSUCIALY-MENDIVIL1, BORIS VOIGT4, FRANTISEK BALUSKA4 and DIEDRIK MENZEL4

1 Departamento de Biología Molecular de Plantas and 3 Unidad de Microscopía del Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México, 2 Universität Tübingen, Entwicklungsgenetik, Tübingen, Germany and 4 Institute of Cellular and Molecular Botany, University of Bonn, Bonn, Germany

* For correspondence. E-mail jdubrov{at}ibt.unam.mx

Received: 13 July 2005    Returned for revision: 3 August 2005    Accepted: 19 January 2006    Published electronically: 6 March 2006

• 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 Phaseolus 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 metaxylem elements, Casparian bands in the endodermis, 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.

Key words: CLSM, Casparian band, xylem, vacuole biogenesis, lateral root development, root development, neutral red


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