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

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.

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Angers-Loustau A, Rainy J, Wartiovaara K (2007). PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories. BMC Mol Biol 8: 77-81.
Atichart P, Bunnag S, Theerakulpisut P (2007). Agrobacteriummediated transformation of Dendrobium secundum (Bl.) Lindl with antisense ACC oxidase. Asian Journal of Plant Sciences 6: 1065-1071.
Chai ML, Xu CJ, Senthil KK, Kim JY, Kim DH (2002). Stable transformation of protocorm-like bodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens. Sci HorticAmsterdam 96: 213-224.
Chin DP, Mishiba K, Mii M (2007). Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium. Plant Cell Rep 26: 735-743.
Gnasekaran P, Poobathy R, Mahmood M, Samian MR, Subramaniam S (2012). Effect of complex organic additives on improving the growth of PLBs of Vanda Kasems Delight. Australian Journal of Crop Science 6: 1245-1248.
Holton T, Cornish E (1995). Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7: 1071-1083.
Jiang F, Wang J, Jia H, Jia W, Wang H, Xiao M (2013). RNAi-mediated silencing of the flavanone 3-hydroxylase gene and its effect on flavonoid biosynthesis in strawberry fruit. J Plant Growth Regul 32: 182-190.
Katsumoto Y, Fukuchi-Mizutani M, Fukui Y, Brugliera F, Holton TA, Karan M, Nakamura N, Yonekura-Sakakibara K, Togami J, Pigeaire A et al. (2007). Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. Plant Cell Physiol 48: 1589-1600.
Khumkarjorn N, Thanonkeo S, Yamada M, Thanonkeo P (2016). Cloning and expression analysis of a flavanone 3-hydroxylase gene in Ascocenda orchid. J Plant Biochem Biot 26: 179-190.
Kuehnle AR, Lewis DH, Markham KR, Mitchell KA, Davies KM, Jordan BR (1997). Floral flavonoids and pH in Dendrobium orchid species and hybrids. Euphytica 95: 187-194.
Kumar A, Singh B, Singh K (2015). Functional characterization of flavanone 3-hydroxylase gene from Phyllanthus emblica (L.). J Plant Biochem Biot 24: 453-460.
Liau CH, You SJ, Prasad V, Hsiao HH, Lu JC, Yang NS, Chan MT (2003). Agrobacterium tumefaciens-mediated transformation of an Oncidium orchid. Plant Cell Rep 21: 993-998.
Mahajan M, Yadav SK (2014). Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco. Plant Mol Biol 85: 551- 573.
Men S, Ming X, Liu R, Wei C, Li Y (2003). Agrobacterium-mediated genetic transformation of a Dendrobium orchid. Plant Cell Tiss Org 75: 63-71.
Meng C, Zhang S, Deng YS, Wang GD, Kong FY (2015). Overexpression of a tomato flavanone 3-hydroxylase-like protein gene improves chilling tolerance in tobacco. Plant Physiol Bioch 96: 388-400.
Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K, Jinbo T, Kimura T (2007). Development of series of Gateway binary vector, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104: 34-41.
Nishihara M, Yamada E, Saito M, Fujita K, Takahashi H, Nakatsuka T (2014). Molecular characterization of mutation in whiteflowered torenia plants. BMC Plant Biol 14: 86-97.
Phlaetita W, Chin DP, Tokuhara K, Nakamura I, Mii M (2015). Agrobacterium-mediated transformation of protocorm-like bodies in Dendrobium Formidible Ugusu. Plant Biotechnol 32: 225-231.
Prescott AG, John P (1996). Dioxygenases: molecular structure and role in plant metabolism. Annu Rev Plant Phys 47: 245-271. Quintanilla-Quintero S, Ortiz P, Bernal J, Gomez A (2011).
Phylogenetic relationships among genera of the subtribe Oncidiinae (Epidendroideae: Orchidaceae) and a new genus: Santanderella. Phytologia 93: 388-406.
Ren W, Zhao L, Zhang L, Wang Y, Cui L, Tang Y, Sun X, Tang K (2011). Molecular cloning and characterization of 4-hydroxyphenylpyruvate dioxygenase gene from Lactuca sativa. J Plant Physiol 168: 1076-1083.
Saiprasad GVS, Raghuveer P, Khetarpal S, Chandra R (2004). Effect of various polyamines on production of protocorm-like bodies in orchid-Dendrobium Sonia. Sci Hortic-Amsterdam 100: 161- 168.
Schöb H, Kunz C, Meins F Jr (1997). Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing. Mol Genet Genomics 256: 581-585.
Schwimmer S, Bevenue A, Weston WJ, Potter AL (1954). Potato composition, survey of major and minor sugar and starch components of white potato. J Agr Food Chem 2: 1284-1290.
Shrestha BR, Chin DP, Tokuhara K, Mii M (2010). Agrobacteriummediated transformation of Vanda using protocorm-like bodies. Asia-Pac J Mol Biol 18: 225-228.
Song X, Diao J, Ji J, Wang G, Guan C, Jin C, Wang Y (2016). Molecular cloning and identification of a flavanone 3-hydroxylase gene from Lycium chinense, and its overexpression enhances drought stress in tobacco. Plant Physiol Bioch 98: 89-100.
Tanaka Y, Brugliera F, Chandler S (2009). Recent progress of flower colour modification by biotechnology. Int J Mol Sci 10: 5350- 5369.
Tanaka Y, Sasaki N, Ohmiya A (2008). Biosynthesis of plant pigment: anthocyanins, betalains and carotenoids. Plant J 54: 733-749.
Tanaka Y, Tsuda S, Kusumi T (1998). Metabolic engineering to modify flower color. Plant Cell Physiol 39: 1119-1126.
Tu Y, Liu F, Guo D, Fan L, Zhu Z, Xue Y, Gao Y, Guo M (2016). Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation. BMC Plant Biol 16: 132.
Udomdee W, Wen P, Lee C, Chin S, Chen F (2014). Effect of sucrose concentration and seed maturity on in vitro germination of Dendrobium nobile hybrids. Plant Growth Regul 72: 249-255. Vacin EF, Went FW (1949). Some pH changes in nutrient solutions. Bot Gaz 110: 605-613. Xiong S, Tian N, Long J, Chen Y, Qin Y, Feng J, Xiao W, Liu S (2016). Molecular cloning and characterization of a flavanone 3-hydroxylase gene from Artemisia annua L. Plant Physiol Bioch 105: 29-36. Yasmin A, Debener T (2010). Transient gene expression in rose petal via Agrobacterium infiltration. Plant Cell Tiss Org 102: 245- 250. Yong JWH, Ge L, Ng YF, Tan SN (2009). The chemical composition and biological properties of coconut (Cocos nucifera L.) water. Molecules 14: 5144-5164.
Zhang L, Chin DP, Mii M (2010). Agrobacterium-mediated transformation of protocorm-like bodies in Cattleya. Plant Cell Tiss Org 103: 41-47.
Zhang Z, Kou X, Fugal K, McLaughlin J (2004). Comparison of HPLC methods for determination of anthocyanins and anthocyanidins in bilberry extracts. J Agr Food Chem 52: 688- 691.
Zuker A, Tzfira T, Ben-Meir H, Ovadis M, Shklarman E, Itzhaki H, Forkmann G, Martens S, Neta-Sharir I, Weiss D et al. (2002). Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene. Mol Breeding 9: 33-41.