Use of Green Fluorescent Protein to Detect Transformed Shoots

Abstract

Visible reporter genes are a very useful approach to evaluating the efficiency of newly developed protocols in plant transformation technology. The basic idea is to distinguish transformed regenerants (shoots or somatic embryos) in an heterogeneous population from untransformed ones without a reduction in the viability of the regeneration tissue, as is the case with selection markers (i.e. antibiotics, herbicides, etc.). Until recently, the reporter genes used for this approach were β-glucuronidase (uidA) and luciferase (Luc) (Ow et al. 1986; Jefferson et al. 1987). Both of these detection procedures require exogenous substrates to visualize the enzymatic activity of the gene products, which in the case of β-glucuronidase (GUS) is destructive for tissues because of the toxicity of the substrate (Jefferson et al. 1987). Furthermore, fixation of the tissue is often required for histochemical detection assays. Detection of luciferase (LUC) expression is possible in vivo, but an exogenous substrate, luciferin, must be applied. Furthermore, the light level generated by the luciferin/ luciferase system is low, and a powerful and expensive low-light detection camera is needed for in vivo detection. Alternatively, enzyme activity can be measured in crude extracts for both marker systems.