A whole-leaf staining and clearing procedure for analysing cytological aspects of interaction between rice plant and rice blast fungus.

Abstract

In order to analyse the cytological aspects of interaction between rice plant and blast fungus, a new whole-leaf staining and clearing procedure has been developed, which includes following treatments of the infected tissue as bathing at 95 C in sodium chlorite solution acidified with acetic acid, dehydrating in alcohol series, staining in autoclave at 121 C with aniline blue dissolved in alcoholic lactophenol and clearing with lactophenol. In a comparative study with present procedure, acidic dyes. especially aniline blue, were found to be much more effective than basic ones to stain the infection hyphae of rice blast fungus in epidermal cells of cleared rice leaves. Acetic acid was better than hydrochloric acid for acidfying rice leaf tissue. Critical proportions of ethanol in staining solution contributed to the staining of infection hyphae and prevented rice leaf tissue from collapse by autoclaving. Staining in autoclave was effective and shortened staining time. With such superiority, this staining and clearing procedure allowed infection hyphae, even those beneath appressoria, to be easily observed and kept infected rice leaf tissue microscopically intact. This procedure should be adaptable for exactly investigating the infection behavior of rice blast fungus and the cellular reactions occurred in rice leaves during infection.