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Spike‐dip transformation of Setaria viridis
Author(s) -
Saha Prasenjit,
Blumwald Eduardo
Publication year - 2016
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13148
Subject(s) - agrobacterium , agrobacterium tumefaciens , transformation (genetics) , biology , green fluorescent protein , transformation efficiency , gus reporter system , reporter gene , setaria viridis , microbiology and biotechnology , transgene , gene , genetically modified crops , acetosyringone , cauliflower mosaic virus , genetics , botany , gene expression , weed
Summary Traditional method of Agrobacterium ‐mediated transformation through the generation of tissue culture had limited success for Setaria viridis , an emerging C4 monocot model. Here we present an efficient in planta method for Agrobacterium ‐mediated genetic transformation of S. viridis using spike dip. Pre‐anthesis developing spikes were dipped into a solution of Agrobacterium tumefaciens strain AGL 1 harboring the β‐glucuronidase ( GUS ) reporter gene driven by the cauliflower mosaic virus 35S (Ca MV 35S) promoter to standardize and optimize conditions for transient as well as stable transformations. A transformation efficiency of 0.8 ± 0.1% was obtained after dipping of 5‐day‐old S3 spikes for 20 min in Agrobacterium cultures containing S. viridis spike‐dip medium supplemented with 0.025% Silwet L‐77 and 200 μ m acetosyringone. Reproducibility of this method was demonstrated by generating stable transgenic lines expressing β‐glucuronidase plus ( GUS plus), green fluorescent protein ( GFP ) and Discosoma sp. red fluorescent protein (DsRed) reporter genes driven by either Ca MV 35S or intron‐interrupted maize ubiquitin (Ubi) promoters from three S. viridis genotypes. Expression of these reporter genes in transient assays as well as in T1 stable transformed plants was monitored using histochemical, fluorometric GUS activity and fluorescence microscopy. Molecular analysis of transgenic lines revealed stable integration of transgenes into the genome, and inherited transgenes expressed in the subsequent generations. This approach provides opportunities for the high‐throughput transformation and potentially facilitates translational research in a monocot model plant.