Improving the Agrobacterium-Mediated Transformation Protocol for Brachypodium Distachyon

Brachypodium distachyon is a grass species which is closely related to the major cereal crops. It possesses many features which are experimentally advantageous, such as a small stature, a short life cycle of 11-18 weeks, and a small genome size (300 Mb) which is fully sequenced. In order to perform genetic research in B. distachyon it is crucial to have an easy and efficient transformation protocol as a routine tool for molecular biology research. Here, we report an improvement on the B. distachyon transformation protocol for the community standard line Bd21 (Alves et al. 2009). In the past, immature embryos up to 0.3 mm in diameter were used to produce compact embryogenic callus (CEC), and transgenic calli were selected, on hygromycin-containing medium, for 6 weeks. In this procedure, immature embryos ranged from 0.3 mm to 0.5 mm in diameter, and the duration of the selection period was shortened by 3 weeks. The CEC proliferation rates of up to 0.3 mm, and 0.3-0.5 mm embryos were 153% and 250%, respectively; and the duration of the hygromycin selection periods of 6 weeks and 3 weeks showed almost comparable transformation efficiencies, 3.5% and 3.7%, respectively. These results suggest that immature embryos of up to 0.5 mm in diameter can be used as explants to induce calli formation, and that 3 weeks of hygromycin selection are sufficient to obtain transgenic material; which optimizes the use of Bd21 donor plants, and significantly reduces the duration of the transformation protocol.