Biosafety of transient transformation technique to express heterologous proteins in tobacco plants

RUBIO, CECILIA, ALVAERZ, MA Y MARCONI PL

Congreso; VIII Encuentro Latinoamericano y del Caribe de Biotecnología - REDBIO ARGENTINA 2013 y IX Simposio Nacional REDBIO; 2013

Using transient transformation technique in plant tissue is a good alternative for the production of recombinant proteins, allowing high levels of expression in a short period of time. Unlike the stable transformation process, this method does not imply any modification in the genome and therefore the plant is not considered a GMO. This method usually implies the use of Agrobacterium tumefaciens, which is covered by the currently existing legislation and risk assessment as Genetically Modified Microorganism (GMM). In order to assess biosafety of transient transformation process when A. tumefaciens for the agroinfiltration of tobacco plants an strain containing an expression vector encoding for green fluorescent protein (GFP) and antibiotic resistance was used. The fluorescence was analysed with an optical microscope (Nikon Eclipse E600) with mercury lamp and appropriated filter. Microbiological and molecular techniques such as GFP-image analyser, PCR, sequencing and RT-PCR were lead to analyze the traceability of GFP during 15 days post-infection. The fluorescence revealed high expression in the agroinfiltration area during 7 days post-infection. The distal areas of injection not showed significant expression of GFP after 7th days post-infection. The results suggest that transient transformation is a highly safe technique so far 9th to 10th days post-infection