Microscopic detection of chloroplast transgenic plastids using fluorescent probe

Shahid Nazir, Muhammad Sarwar Khan

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. 


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References


Pawar BD, Jadhav AS, Kale AA, Chimote VP. Strategies for Use of Environment Friendly Alternative Markers for Detection of Transformation Events in Transgenic Plants. Asian Journal of Experimental Biological Sciences, Spl (2010); 1-5.

Miki B, Mchugh S. Selectable marker genes in transgenic plants: applications, alternatives and biosafety. Journal of Biotechnology, (2004); 107(3): 193-232.

Kaeppler HF, Menon GK, Skadsen RW, Nuutila AM, Carlos AR. Transgenic oat plants via visual selection of cell expressing green fluorescent protein. Plant Cell Reports, (2002); 19(7): 661-666.

Hansch R, Koprek T, Mendel RR, Shulze J. An improved protocol for eliminating endogenous β-glucuronidase background in barley. Plant Science, (1995): 105(1): 63–69.

Haseloff J, Amos B. GFP in plants. Trends in Genetics, (1995); 11(8): 328–9.

Harper BK, Mabon SA, Leffel SM, Halfhill MD, Richards HA, Moyer KA, Stewart CN. Green fluorescent protein as a marker for expression of a second gene in transgenic plants. Nature Biotechnology, (1999); 17(11): 1125-1129.

Jordan MC. Green fluorescent protein as a visual marker for wheat transformation. Plant Cell Reports, (2000); 19(11): 1069-1075.

Daniell H, Khan MS, Allison L. Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends in Plant Science, (2002); 7(2): 84–91.

Nazir S, Khan MS. Chloroplast-encoded chlB gene from Pinus thunbergii promotes root and early chlorophyll pigment development in Nicotiana tabaccum. Molecular Biology Reports, (2012); 39(12): 10637-10646.

Svab Z, Maliga P. High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proceeding of the National Academy of Sciences United Stated of America, (1993); 90(3): 913–917.

Nazir S, Khan MS. Integration of novel chlorophyll genes from black pine into the chloroplast genome of tobacco. Pakistan Journal of Botany, (2013); 45(S1): 595-600.

Khan MS, Hameed W, Nozoe M, Shiina T. Disruption of the psbA gene by the copy correction mechanism reveals that the expression of plastid-encoded genes is regulated by photosynthesis activity. Journal of Plant Research, (2007); 120(3): 421–430.

Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, (1962); 15(3): 473–497.

Khan MS, Maliga P. Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants. Nature Biotechnology, (1999); 17(9): 910–915.

Stewart CN. The utility of green fluorescent protein in transgenic plants. Plant Cell Reports, (2001); 20(5): 376–382.

El-Shemy H, Khalafalla A, Fujita MMK, Ishimoto M. Improvement of protein quality in transgenic soybean plants. Biologia Plantarum, (2007); 51(2): 277–284.

Stewart CN. Monitoring the presence and expression of transgenes in living plants. Trends in Plant Science, (2005); 10(8): 390-396.

El-Shemy H, Mutasim A, Khalafalla M, Ishimoto M. The Role of Green Fluorescent Protein (GFP) in Transgenic Plants to Reduce Gene Silencing Phenomena. Current Issues in Molecular Biology, (2008); 11(1): 21–28.

Richards HA, Han CT, Hopkins RG, Failla ML, Ward WW, Stewart CN. Safety assessment of recombinant green fluorescent protein orally administered to weaned rats. The Journal of Nutrition, (2003); 133(6); 1909-1912.




DOI: http://dx.doi.org/10.62940/als.v5i3.556

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