Factors influencing electroporation-mediated gene transfer to Stylosanthes guianensis (Aubl.) Sw. protoplasts

Abstract

In order to develop a high-efficiency and reproducible transformation protocol for Stylosanthes guianensis we assessed the biological and physical parameters affecting plant electroporation protoplasts. Energy input, as combinations of electric field strengths discharged by different capacitors, electroporation buffer and DNA form were evaluated. Transformation efficiency was assayed in vivo as transient reporter gene expression, using the GFP-coding gene mgfp5 driven by a CaMV 35S constitutive promoter. Energy input and electric field strength had a critical influence on transgene expression with higher transformation levels being achieved with 250 V.cm-1 discharged by 900 and 1000 μF capacitors. Linear plasmid DNA, the absence of chloride and the presence of calcium ions also increased transient gene expression, albeit not significantly.