High-efficiency Agrobacterium-mediated transformation of Cryptomeria japonica D. Don by co-cultivation on filter paper wicks followed by meropenem treatment to eliminate Agrobacterium

摘要

To develop a more efficient genetic transformation system for Cryptomeria japonica (Japanese cedar), the present study evaluated the effects of culture support during co-cultivation and the use of β-lactam antibiotics (meropenem and carbenicillin) to eliminate Agrobacterium on the transformation efficiency. The co-cultivation of embryogenic tissues and bacterium on filter paper wicks prevented the excess growth of Agrobacterium compared with that observed on solid medium, leading to an increased number of gene-transferred cells. Meropenem successfully eliminated the bacterium at low concentrations (10?mg?l?1) and had no phytotoxic effect. The transformed tissues were screened based on kanamycin resistance and green fluorescent protein (GFP) fluorescence, and the integration of the transgene into the plantlet genome was confirmed by Southern blotting. We successfully generated a mean of 105.3±9.02 independent transgenic lines per gram of embryogenic tissues using a combination of filter paper wicks and meropenem. The transformation efficiency of the improved method was approximately 30-fold higher than that observed using the conventional method.