Problems in detecting activity of fluorescent reporter genes – case of DsRED and GFP

Abstract

Reporter genes are indispensible tools in plant biotechnology, used to assess transformation efficiency, select transgenic tissues, study promoters, etc. Advantages of fluorescent markers include non-destructive visualization, simplicity of use, non-toxicity, and stability in most cellular contexts. GFP and DsRED are widely used fluorescent reporters and are considered compatible. Unlike animal systems, plant tissues are notoriously known for autofluorescence, spanning most of the visible spectrum. This limits the application of fluorescent proteins in plants and requires special equipment and a set of control samples, to discern autofluorescence from transgene expression. In the course of study of guaianolide biosynthesis in chicory, several vectors were constructed containing DsRED as co-transformation reporter and GFP as a reporter fused to promoters of the studied genes. To observe DsRED and GFP fluorescence in transgenic chicory tissues, we have used: a system for macro visualization consisting of LED light sources and emission filters, fluorescent stereobinocular, fluorescent microscope and confocal microscope. These methods revealed strong green autofluorescence in newly formed roots, calli, lignified cell walls and parenchyma cells. The intensity of autofluorescence was especially high at large magnification and could obstruct GFP visualization. DsRED fluorescence, on the other hand, was easily discerned from any kind of plant autofluorescence. Another problem was signal crossover from DsRED into GFP channel. DsRED has a small emission peak in the green part of the spectrum, originating from its chromophore maturation. Thus it was impossible to separate fluorescent emission of these two markers present in the same tissue without using spectral deconvolution techniques.