A practical protocol for particle bombardment-mediated transformation of Phyllostachys bamboo suspension cells

概要

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We developed a particle bombardment-mediated transformation protocol in Phyllostachys bamboo by optimizing the growth efficiency of a target cell culture system. Under the optimal condition, i.e. Murashige and Skoog medium containing 680 mg l−1 KH2PO4 and 10 μM Picloram, bamboo suspension cells actively proliferated at ca. 80 ml sedimented cell volume per 100 ml medium in 2 weeks. Log phased cells, i.e. 8 to 13-day-old suspension cells, which showed synchronous cell divisions with uniform morphology, were selected for the bombardment. We found that a target distance, 6 cm was much better for the transient gene expression (222 GUS-positive cells/dish/shot in average) than that of 9 cm (38 GUS-positive cells/dish/shot in average) in the target cells. When the bombardment was carried out using lag phased cells, e.g. 5-d-old cells, no or less GUS-positive cells could be seen. A high generation of stable transgenic bamboo cells was achieved with constructs expressing hygromycin phosphotransferase gene and enhanced fluorescent protein genes namely AcGFP1 and mCherry.