Visual assessments of DsRed and GFP expression and co-expression of GFP and DsRed in the same plant at different stages in culture system: A1 to A3, expression of DsRed at callus, embryo and plantlet stages, respectively; B1 to B3, expression of GFP at callus, embryo, and plantlet stages, respectively;C1 to C3, co-expression of DsRed and GFP within the same tissues at callus, embryo, and plantlet stages, respectively, visualized by means of using blue GFP/DAP filter set with 379– 401-nm excitation wavelength, 435–485-nm emission wavelength, TRITC filter with HQ 530–560-nm excitation wavelength and 590–650-nm emission wavelength for DsRed. D1 to D3, images of callus, embryo, and in vitro plantlets respectively under white light.
Marker genes have proved extremely useful for reporting gene expression in transformed plants. The ß-glucuronidase (GUS) gene has been used extensively .Transformed tissues or patterns of gene expression can be identified histochemically, but this is generally a destructive test and is not suitable for assaying primary transformants, nor for following the time course of gene expression in living plants, nor as a means of rapidly screening segregating populations of seedlings. The green fluorescent protein(GFP) from the cnidarian jellyfish Aequorea victoria shares none of these problems, and there has been much interest in using the protein as a genetic marker in transgenic Arabidopsis thaliana.
Aequorea victoria are brightly luminescent, with glowing points around the margin of the jellyfish umbrella. Light arises from yellow tissue masses that each consist of about 6000-7000 photogenic cells. The cytoplasm of these cells is densely packed with fine granules that contain the components necessary for bioluminescence [2, 3]. In other bioluminescent coelenterates these have been characterised as 0.2 micron diameter particles enclosed by a unit membrane, and have been termed lumisomes .
The components required for bioluminescence include a Ca++ activated photoprotein, aequorin, that emits blue-green light, and an accessory green fluorescent protein (GFP),which accepts energy from aequorin and re-emits it as green light . GFP is an extremely stable protein of 238 amino acids . The fluorescent properties of the protein are unaffected by prolonged treatment with 6M guanidine HCl, 8M urea or 1% SDS, and
two day treatment with various proteases such as trypsin, chymotrypsin, papain,subtilisin, thermolysin and pancreatin at concentrations up to 1 mg/ml fail to alter the intensity of GFP fluorescence . GFP is stable in neutral buffers up to 65oC, and displays a broad range of pH stability from 5.5 to 12. The protein is intensely fluorescent,with a quantum efficiency of approximately 80% and molar extinction coefficient of 2.2x104 cm-1 M-1  (after correction for the known molecular weight). GFP fluoresces maximally when excited at 400 nm with a lesser peak at 475 nm, and fluorescence emission peaks at 509nm .
GFP expression in plants
GFP has been successfully expressed at high levels in tobacco plants using the cytoplasmic RNA viruses potato virus X and tobacco mosaic virus. In these experiments, the gene was directly expressed as a viral mRNA in infected cells, and very high levels of GFP fluorescence were seen.
In contrast to the efficient RNA virus-mediated expression of GFP, variable results have been obtained with transformed cells and plants. Although green fluorescence has been seen in gfp transformed protoplasts of citrus and maize , we and others have seen no fluorescence in Arabidopsis. Hu and Cheng have reported that no signal was seen in gfp transformed Arabidopsis thaliana protoplasts. Reichel and colleagues also failed to dectect fluorescence in gfp transformed Arabidopsis, tobacco or barley protoplasts . Sheen and colleagues also saw no expression of a CAB2-driven gfp gene in transgenic Arabidopsis plants , and Pang etal. saw little or no expression in gfp transformed wheat, corn, tobacco and Arabidopsis plants. There appeared to be a need for substantial improvement of expression the wild-type gfp gene in plants.
The production of GFP fluorescence in plants requires that: (i) the GFP apoprotein be produced in suitable amounts within plant cells, and (ii) the non-fluorescent apoprotein undergoes efficient post-translational modification to produce the mature GFP. The high levels of GFP fluorescence seen in plants infected with suitable RNA virus vectors [13, 14] demonstrate that the protein can undergo efficient post-translation maturation in plants. It has now been shown that expression of the gfp is curtailed by aberrant mRNA splicing in Arabidopsis, and alteration of the codon usage of gfp is required to avoid recognition of a cryptic intron, and allow proper expression of the fluorescent protein in this and other plant species.