3.2. Agroinfiltration of the Nicotiana Tabacum Leaves

The competent Agrobacterium tumefaciens strain GV3101 (provided by Dr. Salmanian, National Institute of Genetic Engineering and Biotechnology of Iran was transformed with the recombinant plasmids purified from E. coli using calcium chloride transformation protocol (32) and selected in the presence of 30 µg.mL^-1 kanamycin and 10 µg.mL^-1 gentamicin. The transformations were confirmed by colony PCR. Agrobacterium suspensions were prepared three days prior to the agroinfiltration by growing the bacteria in the liquid Luria-Bertani medium (Merck, Germany) containing antibiotics at 28 °C overnight on a shaker. The fresh medium was inoculated with the O/N culture at a ratio of 1:10 v/v and further incubated for 3 h at 28 °C. Finally, the bacteria were centrifuged, resuspended to an optical density (OD) of 1.0 at 600 nm in the solution of 10 mM MgCl₂, 10 mM MES (pH 5.5) and 2% (w/v) sucrose, and induced for 90 min with 200 μM acetosyringone (Sigma, USA) before the infiltration (33). To the suspension, Tween-20 was added to a final concentration of 0.01% and the suspensions were used for vacuum agroinfiltration.

The young leaves of the tobacco (Nicotiana tabacum var. Samsun provided by Dr. Rajabi Memari, Shahid Chamran University of Ahvaz, Iran) were scratched with a needle and vacuum-agroinfiltrated twice for 2 min at 0.5 mbar. Li-pcambia carrying GFP construct was used to demonstrate the effect of p19 on expression efficiency. The leaves were exposed to the UV light (365 nm) at the day 4 of post infiltration (dpi) using EpiChemi II (EC2) darkroom system from UVP Bioimaging Systems (UVP, Inc.). The Agrobacterium harboring p19 vector was co-infiltrated with Agrobacteria carrying either Hea-Pcambia or Li-Pcambia or mixture to a final OD₆₀₀ of 1.0 for each. The agroinfiltrated leaves were maintained for 4 days at 24 °C with 16 hours of light.