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Agrobacterium-mediated transformation of Dendrobium orchid with the flavanone 3-hydroxylase gene

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The Ascocenda flavanone 3-hydroxylase (AcF3H) gene was successfully transformed into Dendrobium 5N white orchid plants using Agrobacterium-mediated gene transformation. In this study, for the first time, we report the construction of a plant expression vector harboring the AcF3H gene using the Gateway cloning system. | Turkish Journal of Botany Turk J Bot (2017) 41: 442-454 © TÜBİTAK doi:10.3906/bot-1701-13 http://journals.tubitak.gov.tr/botany/ Research Article Agrobacterium-mediated transformation of Dendrobium orchid with the flavanone 3-hydroxylase gene 1 2 3 4,5 4,5, Nuntipa KHUMKARJORN , Sudarat THANONKEO , Mamoru YAMADA , Preekamol KLANRIT , Pornthap THANONKEO * 1 Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen, Thailand 2 Walai Rukhavej Botanical Research Institute, Mahasarakham University, Maha Sarakham, Thailand 3 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan 4 Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen, Thailand 5 Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, Khon Kaen, Thailand Received: 08.01.2017 Accepted/Published Online: 03.04.2017 Final Version: 28.09.2017 Abstract: The Ascocenda flavanone 3-hydroxylase (AcF3H) gene was successfully transformed into Dendrobium 5N white orchid plants using Agrobacterium-mediated gene transformation. In this study, for the first time, we report the construction of a plant expression vector harboring the AcF3H gene using the Gateway cloning system. Protocorm-like bodies (PLBs) were cocultivated with the Agrobacterium tumefaciens strain AGL1 harboring the plant expression vector pGWB5-AcF3H, which contained the hygromycin phosphotransferase (hpt) gene as a selectable marker. The highest transformation efficiency (10.13%) was achieved when PLBs were cocultivated with Agrobacterium cells for 15 min. Three months after the transformation, the plantlets were regenerated, and the transgenic plants were confirmed by polymerase chain reaction (PCR) analysis using specific primers for the hpt gene and 35S promoter region. PCR products of approximately 400 and 500 bp, corresponding to the hpt gene and the 35S promoter, respectively, were detected in the .