Comprehensive Optimization of Agroinfiltration in Transient Expression System in Nicotiana tabacum with RUBY Visible Reporter

Agrobacterium-mediated transient expression is one of the most popular tools in functional genomics and plant biotechnology. However, its efficiency and reproducibility depend on multiple factors. Agroinfiltration conditions were optimized comprehensively to enhance the expression level of betalain biosynthetic genes in the model plant Nicotiana tabacum with the use of the RUBY visual reporter system. The effects of bacterial suspension concentration, infiltration buffer composition, and delivery method were evaluated. The highest expression efficiency was achieved with a suspension of OD600 = 1.0; a buffer containing acetosyringone (50 μM), 2-(N-morpholino)ethanesulfonic acid (MES) (10 mM), sucrose (0.5%), and magnesium chloride (10 mM); and delivery via preliminary leaf micropuncture with a needle roller and subsequent vacuum infiltration. The resulting protocol significantly improves the efficiency of transient expression and can be employed in gene functional analysis, metabolic engineering, and production of biopharmaceutical compounds in plants.