Optimization of an Agrobacterium-mediated transient assay for gene expression studies in anthurium andraeanum

Abstract

Gene function studies in anthurium (Anthurium andraeanum) have been hindered by the low efficiency of stable transformation, the long regeneration time required as well as the genotype-dependent nature of Agrobacterium (Agrobacterium tumemaciens)-mediated transformation protocols. Agrobacterium-mediated transient expression assays can serve as an attractive alternative for investigating gene function once such assays are optimized. The effects of host factors (genotype, explant type, and developmental maturity of explants), Agrobacterium factors (strain, growth phase, and its concentration), media conditions (infiltration medium used, acetosyringone concentration, type of surfactant, and its concentration), and other experimental factors (infiltration time, cocultivation time, and vacuum infiltration) were investigated on the efficiency of Agrobacterium transient transformation, with replications, using transient expression of β-glucuronidase as an indicator. Although the efficiency of transient transformation was initially found to be highly host genotype-dependent, the genotypic differences in transient transformation efficiency diminished as the protocol was optimized. Agrobacterium strain GV3101 grown to an optical density at 600 nM (OD600) of 1.5 and resuspended to a final OD600 of 0.8 in infiltration medium [0.5% glucose, 10 mM 2-(N-morpholino) ethanesulfonic acid] supplemented with 100 μM acetosyringone and 0.05% of a non-ionic surfactant (S240), for an infiltration period of 16 hours and a cocultivation timeframe of 2 days yielded transient transformation efficiencies as high as 100%.