A reliable and high-efficiency Agrobacterium tumefaciens-mediated transformation system of Pogonatherum paniceum embryogenic callus using GFP as a reporter gene

Abstract

Pogonatherum paniceum (Lam.) Hack, a perennial turfgrass, is important in ecological restoration and landscape construction because of its strong root system and attractive appearance. In this paper, we report the establishment of Agrobacterium tumefaciens-mediated transformation of P. paniceum embryogenic callus (EC) using a high-frequency regeneration system. We also optimised the transformation conditions, such as sub-culture and pre-culture of EC, Agrobacterium concentration, infection time, acetosyringone (AS) concentration, co-cultivation duration, bacteriostatic antibiotics, spectinomycin concentration, washing solutions and selection schemes, based on the transformation efficiency evaluated by green fluorescent protein (GFP) visual analysis, callus survival rate and differentiation rate. The optimal solution was prepared under the following conditions: EC was sub-cultured three times and pre-cultured for 4 days prior to transformation, Agrobacterium concentration of OD600 = 0.6, infection time of 10 min, AS concentration of 40 mg/L, co-cultivation duration of 3 days, washing with liquid washing medium plus 250 mg/L cefotaxime, selection with 300 mg/L cefotaxime and 150 mg/L spectinomycin for 2 or 4 weeks of callus selection, 4 or 2 weeks of differentiation selection and 2 weeks of rooting selection. In the optimised transformation system, the transformation efficiency reached nearly 70 %. PCR and liquid Southern hybridization demonstrated the stable integration of the gfp gene into the P. paniceum genome, GFP visual analysis and RT-PCR confirmed gfp gene expression. This reliable and high-efficiency A. tumefaciens-mediated transformation system lays the foundation for genetic improvement and functional gene research of P. paniceum and improves the application of P. paniceum in ecological restoration and landscape construction.