<![CDATA[Agroinfiltration is a genetic transformation method that was developed to transiently express genes in plant cells for the rapid production of plant-based recombinant proteins. Although this method has been widely applied to Nicotiana benthamiana, few studies have used Nicotiana tabacum. To optimize this method in N. tabacum, the Green Fluorescent Protein (GFP) gene was used as a reporter gene to confirm and quantify the efficiency of the transformation. The GFP gene was cloned into the binary vector pRI101AN and the pRI101AN-GFP construct was introduced into plant tissues via the Agrobacterium tumefaciens strain GV3101. Agroinfiltration was carried out on the leaves of one- and two-month-old N. tabacum plants using the needleless syringe infiltration (agroinjection) method. A. tumefaciens was cultured until an optical density (OD₆₀₀) of 1.0, under varying acetosyringone concentrations of 200 μM and 500 μM in MES buffer. Harvesting times were also optimized at 0, 2-, 4-, 6-, and 8-days post-infiltration (dpi). The success of transient gene transfer and expression was shown through the visualization of GFP fluorescence in the infiltrated leaf tissue. The results showed that the addition of 200 μM acetosyringone in MES buffer infiltrated into two-month-old N. tabacum leaves provided the most effective system for agroinfiltration-based transient gene expression, with the highest GFP expression observed at 4 dpi. These findings provide an important basis for the development of plant-based molecular farming platforms, particularly in Indonesia.]]>
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