High-Throughput Transformation for Switchgrass Cell Cultures

Switchgrass (Panicum virgatum), a C4 perennial grass native to North America, is of particular importance due to its potential in the bioenergy field, as well as, the value added products produced from its biomass. Genetic modification through biotechnology provides a fast method to alter cell wall biosynthesis and improve biomass conversion. As such, we are developing a high-throughput transformation system that uses 96-well plates to transform liquid suspension cultures derived from multiple switchgrass genotypes. Our liquid suspension cultures were produced using Type II callus of ‘Alamo’ and ‘Performer’ clones. This callus type was friable and fast-growing and facilitated the production of single cells in our suspension cultures. Additionally, this type of callus has been shown to produce higher transformation efficiencies in switchgrass and other monocots. Suspension cultures were developed using LS-based or MS-based medium supplemented with 0.9 µM 2,4-dichlorophenoxyacetic acid (2,4-D). After 7-10 d in liquid culture, cells were sub-cultured and used for Agrobacterium tumefaciens-mediated transformation. Transformation efficiencies of each cell type were compared using different Agrobacterium densities, temperatures, and acetosyringone concentrations during co-cultivation. Switchgrass transformation efficiencies were compared to those of Nicotiana tabacum ‘Bright Yellow 2’ (BY-2) cells. In this research, transformable cell suspension cultures were established from switchgrass and an efficient, high-throughput transformation protocol was developed for liquid plant cell culture.