Agrobacterium tumefaciens-Mediated Transformation of Setaria viridis

Abstract

Gene transfer methodology, often referred to as transformation, is an important component of a model system research platform. Availability of transformation methods markedly expand the application of a model. We chose to develop an Agrobacterium tumefaciens-mediated gene transfer method for Setaria viridis A10.1. Regenerable callus was recovered from mature seeds without seed coats that were disinfected and cultured on a Murashige and Skoog-based medium supplemented with 40 g/L maltose, 2 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L kinetin, and 4 g/L Gelzan. The gelling agents Gelzan and Phytagel were found to be critical for recovery of a high quality callus as compared to agar that resulted in a gelatinous, brown, non-regenerable callus. For transformation, the callus was infected with the A. tumefaciens strain AGL1 that contained binary vectors with the hygromycin phosphotransferase selectable marker gene, which confers resistance to the antibiotic hygromycin. The transformation efficiency, which is defined as the percent of infected callus that gives rise to at least one independent transgenic line ranged from 0.3 to 15 % depending upon the vector backbone used to design constructs and the gene of interest that was either overexpressed or had a knockdown of expression. Transgenic lines were first verified by PCR, then positive plants were moved forward for copy number determination by either Southern or TaqMan^® analysis. On average, 42 % of the transgenic lines contained one copy of the introduced transgene. Availability of a transformation methodology has contributed to the adoption of S. viridis as a model species by researchers worldwide.