蝴蝶蘭 (Phalaenopsis)擁有吸引人的外觀，同時也是重要的經濟性園藝作物，在蝴蝶蘭花朵中導入香味性狀，能夠增加其商業價值。相較於傳統育種，運用基因工程技術能更快速獲得有香味的蝴蝶蘭，並且不用擔心影響圓整的花形或其他園藝性狀。因此，本研究旨在發展透過穩定可遺傳的基因轉殖以產生具有香味的蝴蝶蘭的方法。前人研究中指出，在大葉蝴蝶蘭 (Phalaenopsis bellina; P. bellina) 測得的揮發性有機化合物 (volatile organic compounds; VOCs) 之中，主要成分為單萜類化合物 (monoterpenes)— 芳樟醇 (linalool) 和香葉醇 (geraniol)，而香葉基焦磷酸合成酶 (geranyl diphosphate synthase; GDPS) 以及萜類合成酶 (terpene synthase; TPS) 是合成單萜類化合物必要的兩個酵素。在此研究中，我們從具香氣的P. bellina中鑑定並選殖了PbGDPS和PbTPS5基因，之後轉殖到沒有香味的蝴蝶蘭P. aphrodite中大量表達目標基因。經過一系列的分析，我們證實了轉殖株#60異源性大量表達了P. bellina的GDPS基因，PbGDPS表達量的增加，可能誘導P. aphrodite內生性的PaTPS5和PaTPS10的表達，因而具有生合成linalool的能力。為了改善並提升轉殖株中linalool的生成量，我們提出了改良策略：在載體的構築中，使用特定在花部表達啟動子序列、同時轉殖PbGDPS和不同的PbTPS增加香氣成分的產生、不在目標基因N端或C端構築螢光蛋白基因，避免影響目標蛋白質的表達。

Phalaenopsis aphrodite (moth orchid) is visually attractive and is an important economic ornamental crop in Taiwan. Producing floral scent in moth orchids can further increase its commercial value. Compare with conventional breeding, the genetic engineering approach is fast and does not change flower shape and its horticultural traits. Therefore, this study aims to develop a scented moth orchid through a stable transformation approach. Previous studies have shown that the main volatile organic compounds (VOCs) of P. bellina are monoterpenes (linalool and geraniol) and geranyl diphosphate synthase (GDPS) and terpene synthase (TPS) are the two essential enzymes for the monoterpenes synthesis. We identified and cloned PbGDPS and PbTPS5 from scented P. bellina and then overexpressed the target genes in scentless moth orchid. We confirmed that the moth orchid transgenic line #60 heterologously overexpressed P. bellina GDPS gene and upregulated PbGDPS contribute to inducing native moth orchid’s PaTPS5 and PaTPS10, hence, may be capable of producing some linalool compounds. To improve linalool production, we suggest that employing a flower-specific promoter and introducing PbGDPS and different PbTPS genes to enhance the production of aromatic compounds. Additionally, avoiding the integration of fluorescent protein genes at the N-terminus or C-terminus of the target gene to prevent interference with the expression of the target protein.

李美娟、陳思吟、蔡瑜卿、安志豪、劉名旂. 2022. 狐狸尾蝶蘭跨屬雜交技術研究. 種苗科技專訊119:9-14.
林晋寬、侯議翔、宮良政. 2008. 台灣蝴蝶蘭產業之技術發展軌跡:專利及品種權分析. 科技發展政策報導5:1-14.
陳世賢. 2007. 台灣蝴蝶蘭產業競爭力分析. 國立臺灣大學高階公共管理組碩士學位論文. 臺北.
陳怡成. 2004. 台灣蝴蝶蘭智慧財產之管理與運用. 台灣花卉園藝202:46-51.
黃靖嵐、李翎竹. 2018. 蝴蝶蘭產業技術需求調查. 農業世界雜誌420:8-19.
楊玉婷. 2010. 全球蘭花發展現況與未來展望植物種苗生技. 農業生技產業季刊21:7-11.
葉志新、李淑真、廖芳心、葉育哲、蔡月夏、蔡媦婷. 2012. 蝴蝶蘭之雜交育種. 2011年花卉研究團隊成果發表會專刊. p. 25-34.
葉志新、廖方心. 2013. 香味蝴蝶蘭育種. 桃園區農業專訊84:2-5.
葉志新、廖芳心、鄭隨和. 2011. 蝴蝶蘭品種染色體數及型態分析. 桃園區農業改良場研究彙報69:47-58.
葉育哲、王進學、羅妙禎、張芝蓉、蔡月夏. 2016. 香氣蝴蝶蘭新品種‘花蓮 1 號-粉蘋果’及其特性. 花蓮區農業改良場研究彙34:1-8.
葉育哲、蔡月夏、施清田、黃鵬. 2014. 香氣蝴蝶蘭品種選育. 農政與農情267期.
趙欣燕. 台灣蝴蝶蘭產業生產型態之研究--以台南地區為例. 2009. 臺灣師範大學地理學系碩士學位論文. 臺北.
劉黃碧圓. 台灣的蝴蝶蘭翩翩起舞. 豐年半月刊53:25-29. 2003.
蕭郁芸. 蝴蝶蘭香氣分泌構造之觀察與香氣組成初探. 2000. 國立成功大學生物學系碩士學位論文. 臺南.
謝廷芳、曾俊弼、戴廷恩. 2016. 蝴蝶蘭品種交易管理之策略與作法. 蝴蝶蘭育種與品種管理策略研討會專刊. p.33-40.
Acri-Nunes-Miranda R, Mondragón-Palomino M. 2014. Expression of paralogous SEP-, FUL-, AG-and STK-like MADS-box genes in wild-type and peloric Phalaenopsis flowers. Frontiers in plant science 5, 76.
Alfieri M, Vaccaro MC, Cappetta E, Ambrosone A, De Tommasi N, Leone A. 2018. Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors. Scientific Reports 8, 11009.
Arditti J. 1992. Fundamentals of orchid biology: John Wiley & Sons.
Atwood JT. 1986. The size of the Orchidaceae and the systematic distribution of epiphytic orchids. Selbyana, 171-186.
Belarmino M, Mii M. 2000. Agrobacterium-mediated genetic transformation of a Phalaenopsis orchid. Plant Cell Reports 19, 435-442.
Bohlmann J, Meyer-Gauen G, Croteau R. 1998. Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proceedings of the National Academy of Sciences 95, 4126-4133.
Chao Y-T, Yen S-H, Yeh J-H, Chen W-C, Shih M-C. 2017. Orchidstra 2.0—A transcriptomics resource for the orchid family. Plant and Cell Physiology 58, e9-e9.
Chase MW, Cameron KM, Freudenstein JV, Pridgeon AM, Salazar G, Van den Berg C, Schuiteman A. 2015. An updated classification of Orchidaceae. Botanical journal of the Linnean Society 177, 151-174.
Chen F, Tholl D, Bohlmann J, Pichersky E. 2011. The family of terpene synthases in plants: a mid‐size family of genes for specialized metabolism that is highly diversified throughout the kingdom. The Plant Journal 66, 212-229.
Chen H-H, Chuang Y-C, Lee M-C. 2021. Diurnal Regulation of the Floral Scent Emission by Light and Circadian Rhythm for Pollinator Attraction in Phalaenopsis Orchids. Orchid Biotechnology Iv: World Scientific, 359-376.
Chen W-H, Kao Y-L, Tang C-Y. 2013. Method for producing polyploid plants of orchids. Google Patents.
Cheon BY, Kim HJ, Oh KH, Bahn SC, Ahn JH, Choi JW, Ok SH, Bae JM, Shin JS. 2004. Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis. Transgenic research 13, 541-549.
Chiou C-Y, Yeh K-W. 2008. Differential expression of MYB gene (OgMYB1) determines color patterning in floral tissue of Oncidium Gower Ramsey. Plant Molecular Biology 66, 379-388.
Chiu Y-T, Chen H-C, Chang C. 2017. The variation of Oncidium Rosy sunset flower volatiles with daily rhythm, flowering period, and flower parts. Molecules 22, 1468.
Christianson DW. 2006. Structural biology and chemistry of the terpenoid cyclases. Chemical reviews 106, 3412-3442.
Christianson DW. 2017. Structural and chemical biology of terpenoid cyclases. Chemical reviews 117, 11570-11648.
Chuang Y-C, Hung Y-C, Tsai W-C, Chen W-H, Chen H-H. 2018. PbbHLH4 regulates floral monoterpene biosynthesis in Phalaenopsis orchids. Journal of experimental botany 69, 4363-4377.
Chuang YC, Lee MC, Chang YL, Chen WH, Chen HH. 2017. Diurnal regulation of the floral scent emission by light and circadian rhythm in the Phalaenopsis orchids. Bot Stud 58, 50.
Colquhoun TA, Verdonk JC, Schimmel BC, Tieman DM, Underwood BA, Clark DG. 2010. Petunia floral volatile benzenoid/phenylpropanoid genes are regulated in a similar manner. Phytochemistry 71, 158-167.
Crane PR, Friis EM, Pedersen KR. 1995. The origin and early diversification of angiosperms. Nature 374, 27-33.
Dressler RL. 1981. The orchids: natural history and classification. (No Title).
Dudareva N, Klempien A, Muhlemann JK, Kaplan I. 2013. Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytologist 198, 16-32.
Dudareva N, Pichersky E, Gershenzon J. 2004. Biochemistry of plant volatiles. Plant physiology 135, 1893-1902.
Fenske MP, Imaizumi T. 2016. Circadian rhythms in floral scent emission. Frontiers in plant science 7, 462.
Fischer MJ, Meyer S, Claudel P, Perrin M, Ginglinger JF, Gertz C, Masson JE, Werck-Reinhardt D, Hugueney P, Karst F. 2013. Specificity of Ocimum basilicum geraniol synthase modified by its expression in different heterologous systems. Journal of biotechnology 163, 24-29.
Flach A, Dondon RC, Singer RB, Koehler S, Amaral MdCE, Marsaioli AJ. 2004. The chemistry of pollination in selected Brazilian Maxillariinae orchids: floral rewards and fragrance. Journal of Chemical Ecology 30, 1045-1056.
Garay LA. 1960. On the origin of the Orchidaceae. Botanical Museum Leaflets, Harvard University 19, 57-96.
Gershenzon J, Dudareva N. 2007. The function of terpene natural products in the natural world. Nature chemical biology 3, 408-414.
Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF. 2000. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant physiology 124, 1854-1865.
Hecker A, Wallmeroth N, Peter S, Blatt MR, Harter K, Grefen C. 2015. Binary 2in1 vectors improve in planta (co) localization and dynamic protein interaction studies. Plant physiology 168, 776-787.
Hsiao Y-Y, Tsai W-C, Kuoh C-S, Huang T-H, Wang H-C, Wu T-S, Leu Y-L, Chen W-H, Chen H-H. 2006. Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway. BMC Plant Biology 6, 1-14.
Hsing H-X, Lin Y-J, Tong C-G, Li M-J, Chen Y-J, Ko S-S. 2016. Efficient and heritable transformation of Phalaenopsis orchids. Botanical studies 57, 1-12.
Hsu C-T, Liao D-C, Wu F-H, Liu N-T, Shen S-C, Chou S-J, Tung S-Y, Yang C-H, Chan M-T, Lin C-S. 2011. Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey. BMC plant biology 11, 1-14.
Huang L-M, Huang H, Chuang Y-C, Chen W-H, Wang C-N, Chen H-H. 2021. Evolution of terpene synthases in Orchidaceae. International journal of molecular sciences 22, 6947.
Jang I-C, Choi W-B, Lee K-H, Song SI, Nahm BH, Kim J-K. 2002. High-level and ubiquitous expression of the rice cytochrome c gene OsCc1 and its promoter activity in transgenic plants provides a useful promoter for transgenesis of monocots. Plant physiology 129, 1473-1481.
Köllner TG, Schnee C, Gershenzon J, Degenhardt J. 2004. The sesquiterpene hydrocarbons of maize (Zea mays) form five groups with distinct developmental and organ-specific distributions. Phytochemistry 65, 1895-1902.
Kamboj D. 2020. Dendrobium and Venda orchids as potential cut flower in North Indian Market. GINMA, Hisar, Haryana.
Kao Y-Y, Chang S-B, Lin T-Y, Hsieh C-H, Chen Y-H, Chen W-H, Chen C-C. 2001. Differential accumulation of heterochromatin as a cause for karyotype variation in Phalaenopsis orchids. Annals of Botany 87, 387-395.
Kolosova N, Gorenstein N, Kish CM, Dudareva N. 2001. Regulation of circadian methyl benzoate emission in diurnally and nocturnally emitting plants. The Plant Cell 13, 2333-2347.
Koyama T, Tajima M, Sano H, Doi T, Koike-Takeshita A, Obata S, Nishino T, Ogura K. 1996. Identification of significant residues in the substrate binding site of Bacillus stearothermophilus farnesyl diphosphate synthase. Biochemistry 35, 9533-9538.
Lee S-H, Li C-W, Liau C-H, Chang P-Y, Liao L-J, Lin C-S, Chan M-T. 2015. Establishment of an Agrobacterium-mediated genetic transformation procedure for the experimental model orchid Erycina pusilla. Plant Cell, Tissue and Organ Culture (PCTOC) 120, 211-220.
Lee Y-I, Chung M-C, Kuo H-C, Wang C-N, Lee Y-C, Lin C-Y, Jiang H, Yeh C-H. 2017. The evolution of genome size and distinct distribution patterns of rDNA in Phalaenopsis (Orchidaceae). Botanical Journal of the Linnean Society 185, 65-80.
Li Y, Dong C, Hu M, Bai Z, Tong C, Zuo R, Liu Y, Cheng X, Cheng M, Huang J. 2019. Identification of flower-specific promoters through comparative transcriptome analysis in Brassica napus. International journal of molecular sciences 20, 5949.
Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. 1998. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis. The Plant Cell 10, 1391-1406.
Lu S, Xu R, Jia J-W, Pang J, Matsuda SP, Chen X-Y. 2002. Cloning and functional characterization of a β-pinene synthase from Artemisia annua that shows a circadian pattern of expression. Plant Physiology 130, 477-486.
McGarvey DJ, Croteau R. 1995. Terpenoid metabolism. The plant cell 7, 1015.
Mirzaee M, Osmani Z, Frébortová J, Frébort I. 2022. Recent advances in molecular farming using monocot plants. Biotechnology Advances 58, 107913.
Mondragón-Palomino M. 2013. Perspectives on MADS-box expression during orchid flower evolution and development. Frontiers in Plant Science 4, 377.
Muhlemann JK, Klempien A, Dudareva N. 2014. Floral volatiles: from biosynthesis to function. Plant, cell & environment 37, 1936-1949.
Muhlemann JK, Waelti MO, Widmer A, Schiestl FP. 2006. Postpollination changes in floral odor in Silene latifolia: adaptive mechanisms for seed-predator avoidance? Journal of chemical ecology 32, 1855-1860.
Mus AA, Gansau JA, Kumar VS, Rusdi NA. 2020. The variation of volatile compounds emitted from aromatic orchid ('Phalaenopsis bellina') at different timing and flowering stages. Plant Omics 13, 78-85.
Nagegowda DA, Gupta P. 2020. Advances in biosynthesis, regulation, and metabolic engineering of plant specialized terpenoids. Plant Science 294, 110457.
Negre F, Kish CM, Boatright J, Underwood B, Shibuya K, Wagner C, Clark DG, Dudareva N. 2003. Regulation of methylbenzoate emission after pollination in snapdragon and petunia flowers. The Plant Cell 15, 2992-3006.
Ogura K, Koyama T. 1998. Enzymatic Aspects of Isoprenoid Chain Elongation. Chemical Reviews 98, 1263-1276.
Paul P, Singh SK, Patra B, Sui X, Pattanaik S, Yuan L. 2017. A differentially regulated AP 2/ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus. New Phytologist 213, 1107-1123.
Pichersky E. 2006. Biology of floral scent: CRC/Taylor & Francis.
Pichersky E, Noel JP, Dudareva N. 2006. Biosynthesis of plant volatiles: nature's diversity and ingenuity. Science 311, 808-811.
Pij Lvd, Dodson CH. 1966. Orchid flowers: their pollination and evolution.
Pino MT, Skinner JS, Park EJ, Jeknić Z, Hayes PM, Thomashow MF, Chen TH. 2007. Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield. Plant Biotechnology Journal 5, 591-604.
Qiao Z, Hu H, Shi S, Yuan X, Yan B, Chen L. 2021. An update on the function, biosynthesis and regulation of floral volatile terpenoids. Horticulturae 7, 451.
Ramya M, Jang S, An H-R, Lee S-Y, Park P-M, Park PH. 2020. Volatile organic compounds from orchids: From synthesis and function to gene regulation. International journal of molecular sciences 21, 1160.
Ramya M, Kwon OK, An HR, Park PM, Baek YS, Park PH. 2017. Floral scent: Regulation and role of MYB transcription factors. Phytochemistry Letters 19, 114-120.
Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K. 2002. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and cold-inducible gene expression. Biochemical and biophysical research communications 290, 998-1009.
Sarker LS, Adal AM, Mahmoud SS. 2020. Diverse transcription factors control monoterpene synthase expression in lavender (Lavandula). Planta 251, 1-5.
Saunders D. 1997. Insect circadian rhythms and photoperiodism. Invertebrate Neuroscience 3, 155-164.
Schiestl FP, Ayasse M, Paulus HF, Löfstedt C, Hansson BS, Ibarra F, Francke W. 1999. Orchid pollination by sexual swindle. Nature 399, 421-421.
Shikata M, Narumi T, Yamaguchi H, Sasaki K, Aida R, Oshima Y, Takiguchi Y, Ohme-Takagi M, Mitsuda N, Ohtsubo N. 2011. Efficient production of novel floral traits in torenia by collective transformation with chimeric repressors of Arabidopsis transcription factors. Plant biotechnology 28, 189-199.
Streatfield SJ. 2007. Approaches to achieve high‐level heterologous protein production in plants. Plant biotechnology journal 5, 2-15.
Underwood BA, Tieman DM, Shibuya K, Dexter RJ, Loucas HM, Simkin AJ, Sims CA, Schmelz EA, Klee HJ, Clark DG. 2005. Ethylene-regulated floral volatile synthesis in petunia corollas. Plant physiology 138, 255-266.
Unsicker SB, Kunert G, Gershenzon J. 2009. Protective perfumes: the role of vegetative volatiles in plant defense against herbivores. Current opinion in plant biology 12, 479-485.
Van Moerkercke A, Steensma P, Gariboldi I, Espoz J, Purnama PC, Schweizer F, Miettinen K, Vanden Bossche R, De Clercq R, Memelink J. 2016. The basic helix‐loop‐helix transcription factor BIS 2 is essential for monoterpenoid indole alkaloid production in the medicinal plant Catharanthus roseus. The Plant Journal 88, 3-12.
Wang KC, Ohnuma S-i. 2000. Isoprenyl diphosphate synthases. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 1529, 33-48.
Whittington DA, Wise ML, Urbansky M, Coates RM, Croteau RB, Christianson DW. 2002. Bornyl diphosphate synthase: structure and strategy for carbocation manipulation by a terpenoid cyclase. Proceedings of the National Academy of Sciences 99, 15375-15380.
Yamaguchi S, Sun T-p, Kawaide H, Kamiya Y. 1998. The GA2 locus of Arabidopsis thaliana encodes ent-kaurene synthase of gibberellin biosynthesis. Plant physiology 116, 1271-1278.
Yeh C-H, Tsai W-Y, Chiang H-M, Wu C-S, Lee Y-I, Lin L-Y, Chen H-C. 2014. Headspace solid-phase microextraction analysis of volatile components in Phalaenopsis Nobby’s Pacific Sunset. Molecules 19, 14080-14093.
Yeh C-W, Zhong H-Q, Ho Y-F, Tian Z-H, Yeh K-W. 2022. The diurnal emission of floral scent in Oncidium hybrid orchid is controlled by CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) through the direct regulation on terpene synthase. BMC Plant Biology 22, 1-12.
Yu H, Yang SH, Goh CJ. 2002. Spatial and temporal expression of the orchid floral homeotic gene DOMADS1 is mediated by its upstream regulatory regions. Plant molecular biology 49, 225-237.
Zhang H, Hou J, Jiang P, Qi S, Xu C, He Q, Ding Z, Wang Z, Zhang K, Li K. 2016. Identification of a 467 bp promoter of maize phosphatidylinositol synthase gene (ZmPIS) which confers high-level gene expression and salinity or osmotic stress inducibility in transgenic tobacco. Frontiers in Plant Science 7, 42.
Zhao C, Yu Z, Silva JATd, He C, Wang H, Si C, Zhang M, Zeng D, Duan J. 2020. Functional characterization of a Dendrobium officinale geraniol synthase DoGES1 involved in floral scent formation. International Journal of Molecular Sciences 21, 7005.
Zhao J, Ren W, Zhi D, Wang L, Xia G. 2007. Arabidopsis DREB1A/CBF3 bestowed transgenic tall fescue increased tolerance to drought stress. Plant Cell Reports 26, 1521-1528.