Background

One of the most common methods to detect dead plant tissue is trypan blue staining (Keogh et al., 1980). This diazo dye is also used in histology and medicine to measure tissue viability through allowing the visualization of cell death¹ (Keogh et al., 1980; Cooksey, 2014). Most microscopic procedures involving trypan blue staining require a long subsequent clearing step using chloral hydrate (CHL), a small organic compound currently used such as a carcinogen, an anesthetic and an analgesic in laboratory animals (Keogh et al., 1980; Lu and Greco, 2006; Salmon et al., 1995). CHL is not approved by the FDA in the USA or the EMA in the European Union for any medical indication (http://www.accessdata.fda.gov/). Only 250 mg or 50 mg of choral hydrate are sufficient to produce adult or pediatric sedation respectively, and its toxicity has also been measured in neonatals (http://www.drugs.com, Salazar et al., 2009). The LD50 (median lethal dose) for an adult is estimated to be a 4-h exposure to 0,440 mg/L vapour concentration, which is also the duration currently recommended for de-staining of plant leaves at a concentration of 250 g/100 ml. Long-term use of chloral hydrate also results in a rapid development of tolerance to its effects and possible addiction, as well as adverse effects including rashes, gastric discomfort and severe kidney, heart, and liver failure (Gelder et al., 2005).

Through avoiding the use of CHL, this protocol allows researchers to stain for plant cell death with trypan blue more rapidly and safely, substantially reducing the risk to researchers. Here we demonstrate the utility of this method by monitoring the course of infection of Col-0 leaves with Botrytis cinerea (B.c), the second phytopathogen fungus on scientific/economic importance with a broad host range, and a high capacity to produce hydrogen peroxide in plants (Rolke et al., 2004; Dean et al., 2012; Lehmann et al., 2015). This protocol has been also applied successfully to other Arabidopsis accessions.
¹Note: Trypan blue is a synthetic compound derived from toluidine, invented by Paul Ehrlich, winner of the Nobel prize in Physiology and Medicine, 1904 (http://www.pei.de/).