The direct and indirect transformation methods on expressing a recombinant Dermaseptin peptide in tobacco transgenic hairy root clones

Abstract

Despite the increasing demand for production of recombinant proteins, only plant cell cultures are considered as the efficient systems for producing eukaryotic valuable products. Although plant cells are grown in the straightforward and simple conditions, optimization of culture conditions is crucial for a higher yield. In the present study, the establishment of transgenic tobacco hairy roots (HRs) expressing a Dermaseptin B1 recombinant antimicrobial peptide (C2-B1) is examined. Direct and indirect transformation (DT and IT) methods were applied to compare the amount of the recombinant peptide production. The integration, transcription and expression of the transgene in the HRs were confirmed by PCR, semi-quantitative RT-PCR and western blot analysis, respectively. The C2-B1 level was quantified by ELISA analysis of HRs extracts. The results of this study showed that the longer the inoculation and co-culture period, the higher the transformation efficiency. Antimicrobial activity by of crude protein extract from transgenic HR clones showed a significant (P ≤ 0.01) difference between HRs derived from DT and IT methodes methodsmethodes . Transgenic clones derived from both DT and IT methodes and grown in liquid medium showed a difference in terms of total protein content significantly. HRs derived from IT methodmethod produced a significantly higher amount of C2-B1 recombinant peptide in liquid B5 medium supplemented with sucrose.