Agrobacterium-mediated genetic transformation of Miscanthus sinensis

Abstract

Miscanthus species are tall perennial rhizomatous grasses with C4 photosynthesis originating from East Asia, and they are considered as important bioenergy crops for biomass production. In this study, Agrobacterium-mediated transformation system for M. sinensis was developed using embryogenic calli derived from mature seeds. In order to establish a stable system, optimum conditions to obtain highly regenerable and transformation-competent embryogenic calli were investigated, and embryogenic calli were efficiently induced with callus induction medium containing 3 mg L−1 2,4-dichlorophenoxyacetic acid and 25 mM l-proline, at pH 5.7 with an induction temperature of 28 °C. In addition, the embryogenic callus induction and regeneration potentials were compared between seven M. sinensis germplasms collected from several sites in Korea, which revealed that the germplasm SNU-M-045 had superior embryogenic callus induction and regeneration potentials. With this germplasm, the genetic transformation of M. sinensis was performed using Agrobacterium tumefaciens EHA105 carrying pCAMBIA1300 with a green fluorescence protein gene as a reporter. After putative transgenic plants were obtained, the genomic integration of transgenes was confirmed by genomic PCR, transgene expression was validated by Northern blot analysis, and the number of transgene integration was confirmed by DNA gel blot analysis. Furthermore, the Agrobacterium-mediated transformation of M. sinensis was also performed with pCAMBIA3301 which contains an herbicide resistance gene (BAR), and we obtained transgenic M. sinensis plants whose herbicide resistance was confirmed by spraying with BASTA®. Therefore, we have established a stable Agrobacterium-mediated transformation system for M. sinensis, and also successfully produced herbicide-resistant Miscanthus plants by introducing BAR gene via the established method.