Microscopic detection of chloroplast transgenic plastids using fluorescent probe

Abstract

Background: Fluorescent marker genes have modernized many areas of molecular biology, specifically in plant biotechnology and genetic engineering studies. The use of fluorescent proteins permits the scientists to purify the desired clones visually in transformation work. Green fluorescent protein (gfp) derived from Aequorea victoria has been the most common and favorite fluorescent marker which is being widely used as a visual selection marker gene. It can be easily visualized under UV light without the involvement of any substrate and is non-destructive as well. Method: A species-specific chloroplast transformation vector was constructed with gfp as a fluorescent marker gene. The recombinant vector was biolistically integrated in tobacco plastome and transgenic cells were initially screened on spectinomycin containing regeneration medium. Results: The successful plastome integration was verified by using cellular DNA from drug resistant clones in PCR and southern blotting. The expression of gfp in transplastomic clones was microscopically investigated using simple florescent as well as confocal laser scanning microscopes. Conclusion: Regeneration of transgenic plants was significantly helped by visual identification of fluorescent at different stages of development, also enabling to identify the homozygous and heterozygous tissues. No toxic effect of the gfp was observed and lack of toxicity as maintained by normal phenotypic performance of plants.

Keywords

• Fluorescent marker
• Microscopy
• Chloroplast transformation
• Tobacco