Expression of CoQ10-producing ddsA transgene by efficient Agrobacterium-mediated transformation in Panicum meyerianum
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Panicum meyerianum Nees is a wild relative of Panicum maximum Jacq. (guinea grass), which is an important warm-season forage grass and biomass crop. We investigated the conditions that maximized the transformation efficiency of P. meyerianum by Agrobacterium infection by monitoring the expression of the β-glucuronidase (GUS) gene. The highest activities of GUS in calli were achieved by the co-cultivation of plants with Agrobacterium at 28°C for 6 days. We transferred the ddsA gene, which encodes decaprenyl diphosphate synthase and is required for coenzyme Q10 (CoQ10) synthesis, into P. meyerianum by using our optimized co-cultivation procedure for transformation. We confirmed by PCR and DNA gel blot hybridization that all hygromycin-resistant plants retained stable insertion of the hpt and ddsA genes. We also demonstrated strong expression of S14:DdsA protein in the leaves of transgenic P. meyerianum. Furthermore, we showed that transgenic P. meyerianum produced CoQ10 at levels 11–20 times higher than that of non-transformants. By comparison, the CoQ9 level in transgenic plants was dramatically reduced. This is the first report of efficient Agrobacterium-mediated transfer of a foreign gene into the warm-season grass P. meyerianum.
KeywordsAgrobacterium-mediated transformation Panicum meyerianum Co-cultivation Coenzyme Q10
Decaprenyl diphosphate synthase gene
Hygromycin phosphotransferase gene
Polymerase chain reaction
We thank Dr. Masumi Ebina for valuable suggestions and Dr. Hiroki Matsuyama for support with the HPLC analysis (National Institute of Livestock and Grassland Science). Also, we thank Taiji Adachi (Osaka, Japan) for critical reading of the manuscript. This work was funded by the Ministry of Agriculture, Forestry and Fisheries (MAFF) research project ‘Development of innovative crops through the molecular analysis of useful genes’.
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