Authors: NUNTIPA KHUMKARJORN, SUDARAT THANONKEO, MAMORU YAMADA, PREEKAMOL KLANRIT, PORNTHAP THANONKEO
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 transgenic plants, while no such product was observed in nontransgenic plants, indicating that the AcF3H gene was integrated into the genome of Dendrobium 5N white orchid plants. The transient expression of the AcF3H gene in Dendrobium 5N white and Dendrobium Anna petals was performed using the agroinfiltration technique, and the results demonstrated that no cyanidin content was detected in the Dendrobium 5N white petals after AcF3H infiltration. In contrast, the cyanidin content was increased by approximately 6% in the Dendrobium Anna petals, suggesting that the AcF3H gene was transiently expressed in this orchid.
Keywords: Flavanone 3-hydroxylase, Dendrobium orchid, Agrobacterium tumefaciens, protocorm-like bodies
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