Improved demethylation in ecological epigenetic experiments: testing a simple and harmless foliar demethylation application

1. Experimental demethylation of plant DNA enables testing for epigenetic effects in a simple and straightforward way without the use of expensive and laborious DNA sequencing. Plants are commonly demethylated during their germination with the application of agents such as 5-azacytidine (5-azaC). However, this approach can cause unwanted effects such as underdeveloped root systems and high mortality of treated plants, hindering a full comparison with untreated plants, and can be applied only on plant reproducing by seeds. Here we test a simple alternative method of plant demethylation, designed to overcome the shortcomings of the germinating method. 2. We compared a novel method of demethylating plants, based on periodical spraying of 5-azaC aqueous solution on established seedlings, with the previous method in which seeds were germinated directly in 5-azaC solution. We quantified the amount of methylated DNA and measured various aspects of plant performance. Also, we demonstrated its a...