Use of the green fluorescent protein to locate α-amylase gene expression in barley grains.

A green fluorescent protein (GFP) gene was cloned between the promoter and 3´ regions from a barley high isoelectric point (pI) α-amylase gene, then inserted into barley. GFP fluorescence was used to locate and quantify expression of the transgene in barley grains following hydration. Light and confocal laser microscopy revealed fluorescence in the known regions of α-amylase synthesis in the scutellar epithelium, aleurone layer and embryonic axis. Fluorescence was quantified using a simple fluorescence assay, which showed induction of the transgene to mirror the induction of α-amylase in aleurone exposed to gibberellic acid. Expression from the transgene was also shown to be inhibited by abscisic acid, in the same way as expression of endogenous α-amylase genes. Overall, the transgenic grain revealed patterns of α-amylase expression before and after germination, and showed strong potential for further studies investigating both α-amylase production and transport of gibberellin in malting grain.