Green fluorescent protein as a vital marker for non-destructive detection of transformation events in transgenic plants

Abstract

Transformation of plants is a popular tool for modifying various desirable traits. Marker genes, like those encoding for bacterial b-glucuronidase (GUS), firefly luciferase (LUC) or jellyfish green fluorescent protein (GFP) have been shown to be very useful for establishing of efficient transformation protocols. Due to favourable properties such as no need of exogenous substrates and easy visualization, GFP has been found to be superior in to other markers in many cases. However, the use of GFP fluorescence is associated with some obstacles, mostly related to the diminishing of green fluorescence in older tissues, variation in fluorescence levels among different tissues and organs, and occasional interference with other fluorescing compounds in plants. This paper briefly summarizes basic GFP properties and applications, and describes in more detail the contribution of GFP to the establishment, evaluation and improvement of transformation procedures for plants. Moreover, features and possible obstacles associated with monitoring GFP fluorescence are discussed.

Green fluorescent protein as a visual selection marker for coffee transformation

“…In the mature cotyledon leaves and young leaves which developed subsequently, the intensity of green fluorescence was maintained (Figs 2f-h). Similar green fluorescence pattern was also demonstrated in transgenic grape (Dhekney et al 2008), tobacco (Hraska et al 2008) and other plant species transformed with gfp gene (Hraska et al 2006) during various developmental stages.…”

“…Green fluorescent sectors were clearly distinguishable from the yellow and red ones. Increase in fluorescence activity after 15 days of cocultivation was noticed in many plant species, such as onion (Eady et al 2000), sugar beet (Zhang et al 2001) and apple (Hraska et al 2006). The high level of fluorescence 15 days after co-cultivation could be due to the formation of stably transformed calli accumulating higher level of GFP.…”

Visual selection allows immediate identification of transgenic rice calli efficiently accumulating transgene products

“…The ideal selection marker in genetic transformation should be readily expressed, easily visualized and non-toxic for humans and the environment (Hraska et al 2006). Fluorescent proteins such as green fluorescent protein (GFP), whose discovery and development led to the award of the Nobel Prize in Chemistry in 2008, are the most useful visual markers (Shimomura et al 1962).…”

“…Moreover, GFP fluorescence was shown to be an accurate tool for protein quantification and can be used as a quantitative selection marker and an indicator of gene expression (Niwa et al 1999;Richards et al 2003a;Stewart 2005). Visual selection using fluorescent proteins is a powerful tool in cases, where the callus proliferation or regeneration step is inefficient under antibiotic or herbicide selection, because it enables selection and regeneration of transgenic plant cells without any additional selection pressure (Hraska et al 2006;Stewart 2001). In addition, the use of antibiotic resistance markers such as hygromycin phosphotransferase (hpt) has been a subject of public concern, due to the possible deleterious effects on other organisms in the ecosystem caused by the use of antibiotics.…”

“…Visual selection could be one such strategy. To date, visual selection using GFP has been performed successfully in various plants (Hraska et al 2006;Stewart 2001). However, to achieve visual selection without antibiotics, a highly efficient transformation system is required, as transformed cells have to be completely separated from the much larger fraction of untransformed cells (Hraska et al 2006).…”