Isolation of viable protoplasts from the aleurone layers of commercial barley malting varieties

Abstract

The use of aleurone-layer protoplasts as a model system for barley germination research offers several advantages over working with the whole tissue, including the ability to more precisely control the level of hormone exposure during experiments, and to more easily perform cell biology studies using microscopy. However, protoplasts from the cultivar Himalaya have dominated these studies, probably owing to the availability of a suitable isolation protocol tailored to these hull-less grains. Here the development of a protocol to isolate aleurone-layer protoplasts from commercial malting barley cultivars is described. By implementing the protocol, protoplasts were isolated from three commercial Australian varieties and their responses to incubation with gibberellic acid evaluated. Key properties of the protoplasts that make them a good model for the aleurone layer are demonstrated. The soluble proteome of the isolated protoplasts was shown to be highly similar to that of the intact tissue. The cell death response of the protoplasts to treatment with gibberellic acid, which involves increased vacuolization, was visualized as increasingly transparent protoplasts under microscopy. The use of isolated protoplasts offers several applications of great potential to further understand the behaviour of the aleurone layer during germination and thus during the malting process. Copyright © 2016 The Institute of Brewing & Distilling.