Gene delivery into intact plants using the HeliosTM gene gun

Abstract

Particle bombardment is a physical method for cell transformation in which high density, sub-cellular sized particles are accelerated to high velocity to carry DNA/RNA into living cells. It is a versatile technique that can be used for transient expression studies as well as for creating stable transformants. The HeliosTM gene gun uses DNA-coated gold particles that are precipitated on the inner wall of a plastic tube and accelerated by pressurized helium. It differs from previous particle bombardment methods since it operates in ambient pressure and can be used. e.g. in the field. The aim of this study was to optimize parameters for transient expression of gene constructs into arabidopsis, tobacco and birch with the HeliosTM Gene Gun. In order to investigate transient gene delivery, we used constructs containing the constitutively active promoter of the CaMV 35S transcript fused with reporter genes encoding luciferase (LUC) and β-glucuronidase (GUS). Optimization was performed in a step-by-step manner. We found that the most critical parameters were helium pressure, the optimum of which varied between plant species, and the amount of gold. Gold particles with a diameter of 0.6 μm were best for all plant species studied. The optimization procedure helped to increase the expression levels five- to ten-fold.