Direct Transfer and Expression of Human GM-CSF in Tobacco Suspension Cell using Agrobacterium-Mediated Transfer System

Abstract

Direct gene transfer methods were established for cell suspension cultures of tobacco (Nicotiana tabacum cv. Havana) using Agrobacterium-mediated transformation system. Agrobacterium strain LBA4404 containing a binary vector carrying the hGM-CSF gene was introduced into tobacco suspension cells. Cell culture cycle affected transformation efficiency. The cells at 5 days after subculture showed the highest response growing 9.7 transformed cells per plate. Genomic PCR and northern blot analysis demonstrated the integration into cell nuclear genome and the expression of hGM-CSF gene in transgenic tobacco suspension cells. The recombinant hGM-CSF was synthesized by the suspension culture of transgenic cells and secreted to the liquid medium at 150.4 µg 1−1 by day 5, which was detected by ELISA analysis. Cell lines that secreted the hGM-CSF protein into medium were obtained within 2 months. This is faster than previously published technique that necessitate plant regeneration. These results suggest that this method may be used to rapidly produce cell suspension cultures that synthesize useful proteins.