Spike-Dip Transformation Method of Setaria viridis

Abstract

Conventional Agrobacterium-mediated transformation of monocots involves labor-intensive and time-consuming in vitro tissue culture methodology to regenerate T0 plants. To overcome these difficulties, a simple in planta Agrobacterium-mediated genetic transformation method for the emerging monocot model Setaria viridis was developed. Initial standardization of transient and stable transformations was performed using A. tumefaciens strain AGL1 harboring the beta-glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S (CaMV35S) promoter to transform preanthesis developing spikes. The method was further optimized by using A. tumefaciens strain EHA105 carrying beta-glucuronidase plus (GUSplus), green fluorescent protein (GFP), and Discosoma sp. red fluorescent protein (DsRed) reporter genes driven by either CaMV35S or an intron-interrupted maize ubiquitin (Ubi) promoters to develop stable transgenic lines from S. viridis. Dipping of 5-day-old S3 spikes into Agrobacterium cultures containing S. viridis spike-dip medium supplemented with 0.025 % Silwet L-77 and 200 mu M acetosyringone for 20 min produced stable transformants at the rate of 0.8 +/- 0.1 %. Transgenic lines showed stable integration of transgenes into the genome, and inherited transgenes followed the Mendelian segregation pattern and were expressed in subsequent generations. This spike-dip method will facilitate high-throughput translational research in a monocot model.