African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5177

Full Length Research Paper

A modified staining technique for the anatomical observation of mycorrhizal roots of woody trees

  Ana Tztzqui Chávez-Bárcenas1*, Judith Lua-Aldama1, Isaac Alejandro Salmeron-Santiago1, María Blanca Silva-Adame1, Pedro Antonio García-Saucedo1 and Victor Olalde-Portugal2
  1Facultad de Agrobiología “Presidente Juárez”, Universidad Michoacana de San Nicolás de Hidalgo. Paseo Lázaro Cárdenas S/N Esq. Berlín, Col. Viveros, Uruapan, Michoacán, CP 60190, México. 2Departamento de Bioquímica y Biotecnología de Plantas, Centro de Investigación y de Estudios Avanzados-IPN, Unidad Irapuato, Km 9.6 Libramiento Nte. Carr. Irapuato-León, Apdo. Postal 629, 36500, Irapuato, Guanajuato, México.
Email: [email protected], [email protected]

  •  Accepted: 05 July 2013
  •  Published: 12 July 2013

Abstract

 

Modifications to the root staining method described by Phillips and Hayman (1970) were performed in order to develop a clear anatomical visualization of the mycorrhization status in avocado roots. Diverse KOH and H2O2 concentrations and various incubation times during clearing and acidification steps were assayed. The best results were obtained with the following treatment: once dried at 50°C for 12 h, 1 cm length roots were submerged in 15% KOH and autoclaved at 115 kPa during 15 min. The solution was thoroughly washed with tap water, then the roots were decolorized with 3% H2O2 for 15 min at room temperature, rinsed out and re-autoclaved in 15% KOH at 115 kPa for 10 min, followed by a second treatment with 3% H2O2 during 20 min. Subsequently, they were acidified in 1% HCl during 5 min, decanted and stained with tryphan blue 0.05% in acetoglycerol (26.66% acetic acid; 33.33% glycerol) having been autoclaved at 115 kPa during 10 min. Following which, the roots were mounted on slides. Roots from five other woody plant species were also tested for the proposed staining protocols. Arbuscular Mycorrhizal (AM) structures such as hyphae, arbuscules, and vesicles inside the roots of all the species tested were stained and clearly visible under a light microscope.

 

Key words: Arbuscular mycorrhizal fungi, Persea americana, root staining of woody fruit trees.