蘭科植物是顯花植物的最大科之一，且形態具多樣化。基於該科植物原生地廣泛分佈在不同的生態棲地，包含不同植株形態、構造及生理特性，蘭科植物花部顏色、構造與大小及著生花朵數之生殖特性，值得研究其生物學特性及其功能性基因。病毒誘導的基因沉默，是一個反向遺傳學的方法，適合分析研究植物基因之功能，尤其適合從播種到開花生育週期偏長，缺乏遺傳研究資訊的蘭花。本研究使用蕙蘭嵌紋病毒所改造的載體建立誘導蝴蝶蘭基因靜默的效率之最佳操作條件，該病毒載體可有效靜默蝴蝶蘭PeUFGT3、PeMADS5 及PeMADS6基因表達效率達95.8％。擬靜默基因之DNA片段插入載體的最佳長度最小可至78至85 bp。以PeMADS6 基因進行測試接種在已具有明顯可見花苞形態之花序植株，有顯著提升基因靜默效果。此外，直接在葉片注射接種取代在花序軸注射接種，產生相同靜默效率，且節省接種時間及減少對植體傷害。進一步，以上述所獲得之病毒接種條件分析自花發育選殖的126個轉錄因子之表現序列標籤，進行高通量病毒誘導基因靜默。靜默PeMADS1及PebHLH均會導致花朵變小，靜默PeMADS1也導致蕊柱伸長及變寬，而靜默PebHLH造成唇瓣之翼瓣向外側彎曲及改變表皮細胞排列。此外，靜默PeMADS7、PeHB及PeZIP則造成花序最初發育之前三朵花苞敗育，但具有完整發育之各花部器官。另外，同時靜默分屬C群(PeMADS1)及B群(PeMADS6)的MADS box基因，獲得一個組合雙基因靜默所致之異常外表型。因此，藉由病毒誘導基因靜默法證實正常表達PeMADS1及PeMADS6乃能確保蝴蝶蘭唇瓣及蕊柱形態與花器表皮細胞表面角質層的正常發育。本論文亦確認應用病毒誘導基因靜默技術可協助了解不同群MADS轉錄因子之間的相互作用，及闡明它們在蘭花花器形態發育之調控機制。

The Orchidaceae is one of the largest and most diverse families of flowering plants. This family occupies a wide range of ecological habitats and exhibits highly specialized morphological, structural, and physiological characteristics. Thus, orchid reproductive biology is of special interest in terms of floral color, morphology, size and number of flowers. Virus-induced gene silencing (VIGS) is a reverse genetic approach used for functional analysis of plant genes, especially those with long life cycles and having few genetic resources, such as orchids. In this study, the modified Cymbidium mosaic virus was used as a VIGS based vector. To explore the applicability and improve the viral silencing efficiency for demarcated functional genes in Phalaenopsis, this VIGS vector was used for silencing PeUFGT3, PeMADS5, and PeMADS6 and induced significant phenotypes with a silencing efficiency up to 95.8% reduction of target gene expressions. In addition, the size of target gene fragments for inserting into VIGS vectors can be as small as 79-85 bp and can still reach 61.5%-95.8% reduction. The silencing efficiency of PeMADS6 has been increased significantly when the inflorescences of the Phalaenopsis plants contained eight internodes and one visible floral bud used as experimental materials. Furthermore, leaf injection saves time and causes less damage to inflorescences for Agro-infiltration, since there is not any difference between leaf- and inflorescence-injection inoculation on efficiencies of virus infection and silenced PeMADS6. To understand individual transcription factors (TFs) that affect orchid floral morphogenesis and development, we used high-throughput VIGS to silence the expression of 126 floral ESTs that encode TFs and examined their functions during floral morphogenesis in Phalaenopsis. Five TFs were associated with obvious phenotypes. Silencing PeMADS7, PeHB or PebZIP led to abortion of the first 3 floral buds, and silencing PeMADS1 or PebHLH affected floral morphogenesis and reduced floral size. Silencing PeMADS1 caused a homeotic transformation of the column into a petal-like column, whereas silencing PebHLH affected the development of lateral lobes of the lip. Co-silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, produced flowers with the combined characteristics of the silencing of each gene, indicating that the functions of the two MADS-box genes ensured the normal morphogenesis and epidermal cellular types within the lip and column. The co-silencing technique will facilitate studies for unraveling the interactions among different classes of MADS-box genes and elucidating the regulatory mechanisms of floral morphogenesis in orchids.

Amasino, R., 2010. Seasonal and developmental timing of flowering. The Plant Journal 61, 1001-1013.

Ambrose, B.A., Lerner, D.R., Ciceri, P., Padilla, C.M., Yanofsky, M.F., Schmidt, R.J., 2000. Molecular and genetic analyses of the silky1 gene reveal conservation in floral organ specification between eudicots and monocots. Molecular Cell 5, 569-579.

Andres, F., Coupland, G., 2012. The genetic basis of flowering responses to seasonal cues. Nature Reviews Genetics 13, 627-639.

Arora, R., Agarwal, P., Ray, S., Singh, A.K., Singh, V.P., Tyagi, A.K., Kapoor, S., 2007. MADS-box gene family in rice: genome-wide identification, organization and expression profiling during reproductive development and stress. BMC Genomics 8, 242.

Baulcombe, D., 1999. Viruses and gene silencing in plants. Archives of Virology Supplementa Volume 15, 189-201.

Bernacki, S., Karimi, M., Hilson, P., Robertson, N., 2010. Virus-induced gene silencing as a reverse genetics tool to study gene function. Methods in Molecular Biology 655, 27-45.

Bernier, G., Havelange, A., Houssa, C., Petitjean, A., Lejeune, P., 1993. Physiological signals that induce flowering. The Plant Cell 5, 1147-1155.

Bowman, J.L., Smyth, D.R., Meyerowitz, E.M., 1989. Genes directing flower development in Arabidopsis. The Plant Cell 1, 37-52.

Bowman, J.L., Smyth, D.R., Meyerowitz, E.M., 1991. Genetic interactions among floral homeotic genes of Arabidopsis. Development 112, 1-20.

Brigneti, G., Martin-Hernandez, A.M., Jin, H., Chen, J., Baulcombe, D.C., Baker, B., Jones, J.D., 2004. Virus-induced gene silencing in Solanum species. The Plant Journal 39, 264-272.

Burch-Smith, T.M., Schiff, M., Liu, Y., Dinesh-Kumar, S.P., 2006. Efficient virus-induced gene silencing in Arabidopsis. Plant Physiology 142, 21-27.

Chai, Y.M., Jia, H.F., Li, C.L., Dong, Q.H., Shen, Y.Y., 2011. FaPYR1 is involved in strawberry fruit ripening. Journal of Experimental Botany 62, 5079-5089.

Chang, Y.-Y., Chiu, Y.-F., Wu, J.-W., Yang, C.-H., 2009. Four orchid (Oncidium Gower Ramsey) AP1/AGL9-like MADS box genes show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis thaliana. Plant and Cell Physiology 50, 1425-1438.

Chang, Y.Y., Kao, N.H., Li, J.Y., Hsu, W.H., Liang, Y.L., Wu, J.W., Yang, C.H., 2010. Characterization of the possible roles for B class MADS box genes in regulation of perianth formation in orchid. Plant Physiology 152, 837-853.

Chen, D., Guo, B., Hexige, S., Zhang, T., Shen, D., Ming, F., 2007. SQUA-like genes in the orchid Phalaenopsis are expressed in both vegetative and reproductive tissues. Planta 226, 369-380.

Chen, Y.Y., Lee, P.F., Hsiao, Y.Y., Wu, W.L., Pan, Z.J., Lee, Y.I., Liu, K.W., Chen, L.J., Liu, Z.J., Tsai, W.C., 2012. C- and D-class MADS-box genes from Phalaenopsis equestris (Orchidaceae) display functions in gynostemium and ovule development. Plant and Cell Physiology 53, 1053-1067.

Chung, E., Seong, E., Kim, Y.C., Chung, E.J., Oh, S.K., Lee, S., Park, J.M., Joung, Y.H., Choi, D., 2004. A method of high frequency virus-induced gene silencing in chili pepper (Capsicum annuum L. cv. Bukang). Molecules and Cells 17, 377-380.

Chung, Y.Y., Kim, S.R., Finkel, D., Yanofsky, M.F., An, G., 1994. Early flowering and reduced apical dominance result from ectopic expression of a rice MADS box gene. Plant Molecular Biology 26, 657-665.

Chung, Y.Y., Kim, S.R., Kang, H.G., Noh, Y.S., Park, M.C., Finkel, D., An, G., 1995. Characterization of two rice MADS box genes homologous to GLOBOSA. Plant Science 109, 45-56.

Ciaffi, M., Paolacci, A.R., Tanzarella, O.A., Porceddu, E., 2011. Molecular aspects of flower development in grasses. Sexual Plant Reproduction 24, 247-282.

Coen, E.S., Meyerowitz, E.M., 1991. The war of the whorls: genetic interactions controlling flower development. Nature 353, 31-37.

Constantin, G.D., Krath, B.N., MacFarlane, S.A., Nicolaisen, M., Johansen, I.E., Lund, O.S., 2004. Virus-induced gene silencing as a tool for functional genomics in a legume species. The Plant Journal 40, 622-631.

Corbesier, L., Vincent, C., Jang, S., Fornara, F., Fan, Q., Searle, I., Giakountis, A., Farrona, S., Gissot, L., Turnbull, C., Coupland, G., 2007. FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 316, 1030-1033.

De Bodt, S., Raes, J., Van de Peer, Y., Theissen, G., 2003. And then there were many: MADS goes genomic. Trends in Plant Science 8, 475-483.

Di Stilio, V.S., Kumar, R.A., Oddone, A.M., Tolkin, T.R., Salles, P., McCarty, K., 2010. Virus-induced gene silencing as a tool for comparative functional studies in Thalictrum. PLoS One 5, e12064.

Ding, L., Wang, Y., Yu, H., 2013. Overexpression of DOSOC1, an ortholog of Arabidopsis SOC1, promotes flowering in the orchid Dendrobium Chao Parya Smile. Plant and Cell Physiology 54, 595-608.

Ding, X.S., Schneider, W.L., Chaluvadi, S.R., Mian, M.A., Nelson, R.S., 2006. Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts. Molecular Plant-Microbe Interactions 19, 1229-1239.

Ditta, G., Pinyopich, A., Robles, P., Pelaz, S., Yanofsky, M.F., 2004. The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Current Biology 14, 1935-1940.

Doi, K., Izawa, T., Fuse, T., Yamanouchi, U., Kubo, T., Shimatani, Z., Yano, M., Yoshimura, A., 2004. Ehd1, a B-type response regulator in rice, confers short-day promotion of flowering and controls FT-like gene expression independently of Hd1. Genes and Development 18, 926-936.

Dreni, L., Jacchia, S., Fornara, F., Fornari, M., Ouwerkerk, P.B., An, G., Colombo, L., Kater, M.M., 2007. The D-lineage MADS-box gene OsMADS13 controls ovule identity in rice. The Plant Journal 52, 690-699.

Dreni, L., Pilatone, A., Yun, D., Erreni, S., Pajoro, A., Caporali, E., Zhang, D., Kater, M.M., 2011. Functional analysis of all AGAMOUS subfamily members in rice reveals their roles in reproductive organ identity determination and meristem determinacy. The Plant Cell 23, 2850-2863.

Favaro, R., Immink, R.G., Ferioli, V., Bernasconi, B., Byzova, M., Angenent, G.C., Kater, M., Colombo, L., 2002. Ovule-specific MADS-box proteins have conserved protein-protein interactions in monocot and dicot plants. Molecular and General Genetics 268, 152-159.

Favaro, R., Pinyopich, A., Battaglia, R., Kooiker, M., Borghi, L., Ditta, G., Yanofsky, M.F., Kater, M.M., Colombo, L., 2003. MADS-box protein complexes control carpel and ovule development in Arabidopsis. The Plant Cell 15, 2603-2611.

Fornara, F., Panigrahi, K.C., Gissot, L., Sauerbrunn, N., Ruhl, M., Jarillo, J.A., Coupland, G., 2009. Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are essential for a photoperiodic flowering response. Developmental Cell 17, 75-86.

Fornara, F., Parenicova, L., Falasca, G., Pelucchi, N., Masiero, S., Ciannamea, S., Lopez-Dee, Z., Altamura, M.M., Colombo, L., Kater, M.M., 2004. Functional characterization of OsMADS18, a member of the AP1/SQUA subfamily of MADS box genes. Plant Physiology 135, 2207-2219.

Golenberg, E.M., Sather, D.N., Hancock, L.C., Buckley, K.J., Villafranco, N.M., Bisaro, D.M., 2009. Development of a gene silencing DNA vector derived from a broad host range geminivirus. Plant Methods 5, 9.

Goto, K., Meyerowitz, E.M., 1994. Function and regulation of the Arabidopsis floral homeotic gene PISTILLATA. Genes and Development 8, 1548-1560.

Greco, R., Stagi, L., Colombo, L., Angenent, G.C., Sari-Gorla, M., Pe, M.E., 1997. MADS box genes expressed in developing inflorescences of rice and sorghum. Molecular and General Genetics 253, 615-623.

Gutierrez-Cortines, M.E., Davies, B., 2000. Beyond the ABCs: ternary complex formation in the control of floral organ identity. Trends in Plant Science 5, 471-476.

Hileman, L.C., Drea, S., Martino, G., Litt, A., Irish, V.F., 2005. Virus-induced gene silencing is an effective tool for assaying gene function in the basal eudicot species Papaver somniferum (opium poppy). The Plant Journal 44, 334-341.

Honma, T., Goto, K., 2001. Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409, 525-529.
Hou, C.J., Yang, C.H., 2009. Functional analysis of FT and TFL1 orthologs from orchid
(Oncidium Gower Ramsey) that regulate the vegetative to reproductive transition. Plant and Cell Physiology 50, 1544-1557.

Hsiao, Y.Y., Pan, Z.J., Hsu, C.C., Yang, Y.P., Hsu, Y.C., Chuang, Y.C., Shih, H.H., Chen, W.H., Tsai, W.C., Chen, H.H., 2011. Research on orchid biology and biotechnology. Plant and Cell Physiology 52, 1467-1486.

Hsu, H.F., Hsieh, W.P., Chen, M.K., Chang, Y.Y., Yang, C.H., 2010. C/D class MADS box genes from two monocots, orchid (Oncidium Gower Ramsey) and lily (Lilium longiflorum), exhibit different effects on floral transition and formation in Arabidopsis thaliana. Plant and Cell Physiology 51, 1029-1045.

Hsu, H.F., Huang, C.H., Chou, L.T., Yang, C.H., 2003. Ectopic expression of an orchid (Oncidium Gower Ramsey) AGL6-like gene promotes flowering by activating flowering time genes in Arabidopsis thaliana. Plant and Cell Physiology 44, 783-794.

Hsu, H.F., Yang, C.H., 2002. An orchid (Oncidium Gower Ramsey) AP3-like MADS gene regulates floral formation and initiation. Plant and Cell Physiology 43, 1198-1209.

Igarashi, A., Yamagata, K., Sugai, T., Takahashi, Y., Sugawara, E., Tamura, A., Yaegashi, H., Yamagishi, N., Takahashi, T., Isogai, M., Takahashi, H., Yoshikawa, N., 2009. Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes. Virology 386, 407-416.

Immink, R.G., Kaufmann, K., Angenent, G.C., 2010. The 'ABC' of MADS domain protein behaviour and interactions. Seminars in Cell and Developmental Biology 21, 87-93.
Jack, T., Brockman, L.L., Meyerowitz, E.M., 1992. The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell 68, 683-697.

Jack, T., Fox, G.L., Meyerowitz, E.M., 1994. Arabidopsis homeotic gene APETALA3 ectopic expression: transcriptional and posttranscriptional regulation determine floral organ identity. Cell 76, 703-716.

Jeon, J.S., Jang, S., Lee, S., Nam, J., Kim, C., Lee, S.H., Chung, Y.Y., Kim, S.R., Lee, Y.H., Cho, Y.G., An, G., 2000a. leafy hull sterile1 is a homeotic mutation in a rice MADS box gene affecting rice flower development. The Plant Cell 12, 871-884.

Jeon, J.S., Lee, S., Jung, K.H., Yang, W.S., Yi, G.H., Oh, B.G., An, G., 2000b. Production of transgenic rice plants showing reduced heading date and plant height by ectopic expression of rice MADS-box genes. Molecular Breeding 6, 581-592.

Jofuku, K.D., den Boer, B.G., Van Montagu, M., Okamuro, J.K., 1994. Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. The Plant Cell 6, 1211-1225.
Kalantidis, K., Schumacher, H.T., Alexiadis, T., Helm, J.M., 2008. RNA silencing movement in plants. Biol Cell 100, 13-26.

Kammen, A., 1997. Virus-induced gene silencing in infected and transgenic plants. Trends in Plant Science 2, 409-411.

Kang, H.G., An, G., 1997. Isolation and characterization of a rice MADS box gene belonging to the AGL2 gene family. Molecules and Cells 7, 45-51.

Kang, H.G., Jeon, J.S., Lee, S., An, G., 1998. Identification of class B and class C floral organ identity genes from rice plants. Plant Molecular Biology 38, 1021-1029.

Kater, M.M., Dreni, L., Colombo, L., 2006. Functional conservation of MADS-box factors controlling floral organ identity in rice and Arabidopsis. Journal of Experimental Botany 57, 3433-3444.

Kim, H.J., Hyun, Y., Park, J.Y., Park, M.J., Park, M.K., Kim, M.D., Lee, M.H., Moon, J., Lee, I., Kim, J., 2004. A genetic link between cold responses and flowering time through FVE in Arabidopsis thaliana. Nature Genetics 36, 167-171.

Kim, S.L., Lee, S., Kim, H.J., Nam, H.G., An, G., 2007a. OsMADS51 is a short-day flowering promoter that functions upstream of Ehd1, OsMADS14, and Hd3a. Plant Physiology 145, 1484-1494.

Kim, S.Y., Yun, P.Y., Fukuda, T., Ochiai, T., Yokoyama, J., Kameya, T., Kanno, A., 2007b. Expression of a DEFICIENS-like gene correlates with the differentiation between sepal and petal in the orchid, Habenaria radiata (Orchidaceae). Plant and Cell Physiology 172, 319-326.

Kim, W., Ahn, H.J., Chiou, T.J., Ahn, J.H., 2011a. The role of the miR399-PHO2 module in the regulation of flowering time in response to different ambient temperatures in Arabidopsis thaliana. Molecules and Cells 32, 83–88.

Kim, B.M., Inaba, J., Masuta, C., 2011b. Virus induced gene silencing in Antirrhinum majus using the Cucumber mosaic virus vector: functional analysis of the AINTEGUMENTA (Am-ANT) gene of A. majus. Horticulture, Environment, and Biotechnology 52, 176-182.

Kim, J.J., Lee, J.H., Kim, W., Jung, H.S., Huijser. P., Ahn, J.H., 2012. The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis. Plant Physiology 159, 461–478.

Kjemtrup, S., Sampson, K.S., Peele, C.G., Nguyen, L.V., Conkling, M.A., Thompson, W.F., Robertson, D., 1998. Gene silencing from plant DNA carried by a Geminivirus. The Plant Journal 14, 91-100.

Komeda, Y., 2004. Genetic regulation of time to flower in Arabidopsis thaliana. Annual Review of Plant Biology 55, 521-535.

Krizek, B.A., Fletcher, J.C., 2005. Molecular mechanisms of flower development: an armchair guide. Nature Reviews Genetics 6, 688-698.

Krizek, B.A., Meyerowitz, E.M., 1996. The Arabidopsis homeotic genes APETALA3 and
PISTILLATA are sufficient to provide the B class organ identity function. Development 122, 11-22.

Kumagai, M.H., Donson, J., della-Cioppa, G., Harvey, D., Hanley, K., Grill, L.K., 1995. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proceedings of the National Academy of Sciences of the United States of America 92, 1679-1683.

Kunst, L., Klenz, J.E., Martinez-Zapater, J., Haughn, G.W., 1989. AP2 gene determines the identity of perianth organs in flowers of Arabidopsis thaliana. The Plant Cell 1, 1195-1208.

Kyozuka, J., Kobayashi, T., Morita, M., Shimamoto, K., 2000. Spatially and temporally regulated expression of rice MADS box genes with similarity to Arabidopsis class A, B and C genes. Plant and Cell Physiology 41, 710-718.

Kyozuka, J., Shimamoto, K., 2002. Ectopic expression of OsMADS3, a rice ortholog of AGAMOUS, caused a homeotic transformation of lodicules to stamens in transgenic rice plants. Plant and Cell Physiology 43, 130-135.

Lee, S., Jeon, J.S., An, K., Moon, Y.H., Lee, S., Chung, Y.Y., An, G., 2003. Alteration of floral organ identity in rice through ectopic expression of OsMADS16. Planta 217, 904-911.

Lee, H., Yoo, S.J., Lee, J.H., Kim, W., Yoo, S.K., Fitzgerald, H., Carrington, J.C., Ahn, J.H., 2010. Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis. Nucleic Acids Research 38, 3081–3093

Lenhard, M., Bohnert, A., Jurgens, G., Laux, T., 2001. Termination of stem cell maintenance in Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS. Cell 105, 805-814.

Li, D., Behjatnia, S.A., Dry, I.B., Walker, A.R., Randles, J.W., Rezaian, M.A., 2008. Tomato leaf curl virus satellite DNA as a gene silencing vector activated by helper virus infection. Virus Research 136, 30-34.

Li, R.H., Wang, A.K., Sun, S.L., Liang, S., Wang, X.J., Ye, Q.S., Li, H.G., 2012. Functional characterization of FT and MFT ortholog genes in orchid (Dendrobium nobile Lindl) that regulate the vegetative to reproductive transition in Arabidopsis. Plant Cell Tissue and Organ Culture 111, 143-151.

Liscombe, D.K., O'Connor, S.E., 2011. A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus. Phytochemistry 72, 1969-1977.

Liu, Y., Schiff, M., Dinesh-Kumar, S.P., 2002. Virus-induced gene silencing in tomato. The Plant Journal 31, 777-786.

Lu, H.C., Chen, H.H., Tsai, W.C., Chen, W.H., Su, H.J., Chang, D.C., Yeh, H.H., 2007.
Strategies for functional validation of genes involved in reproductive stages of orchids. Plant Physiology 143, 558-569.

Lu, H.C., Hsieh, M.H., Chen, C.E., Chen, H.H., Wang, H.I., Yeh, H.H., 2012a. A high-throughput virus-induced gene-silencing vector for screening transcription factors in virus-induced plant defense response in orchid. Molecular Plant-Microbe Interactions 25, 738-746.

Lu, S.J., Wei, H., Wang, Y., Wang, H.M., Yang, R.F., Zhang, X.b., J.M., T., 2012b. Overexpression of a transcription factor OsMADS15 modifies plant architecture and flowering time in rice (Oryza sativa L.). Plant Molecular Biology Reporter 30, 1461-1469.

Malcomber, S.T., Kellogg, E.A., 2005. SEPALLATA gene diversification: brave new whorls. Trends in Plant Science 10, 427-435.

Mandel, M.A., Gustafson-Brown, C., Savidge, B., Yanofsky, M.F., 1992. Molecular characterization of the Arabidopsis floral homeotic gene APETALA1. Nature 360, 273-277.

Meyerowitz, E.M., Smyth, D.R., Bowman, J.L., 1989. Abnormal flowers and pattern formation in floral development. Development 106, 209-217.

Mizukami, Y., Ma, H., 1992. Ectopic expression of the floral homeotic gene AGAMOUS in transgenic Arabidopsis plants alters floral organ identity. Cell 71, 119-131.

Mondragon-Palomino, M., Hiese, L., Harter, A., Koch, M.A., Theissen, G., 2009. Positive selection and ancient duplications in the evolution of class B floral homeotic genes of orchids and grasses. BMC Evolutionary Biology 9, 81.

Moon, Y.H., Jung, J.Y., Kang, H.G., An, G., 1999. Identification of a rice APETALA3 homologue by yeast two-hybrid screening. Plant Molecular Biology 40, 167-177.

Mutasa-Goettgens, E., Hedden, P., 2009. Gibberellin as a factor in floral regulatory networks. Journal of Experimental Botany 60, 1979-1989.

Nagamatsu, A., Masuta, C., Senda, M., Matsuura, H., Kasai, A., Hong, J.S., Kitamura, K., Abe, J., Kanazawa, A., 2007. Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing. Plant Biotechnology Journal 5, 778-790.

Nagasawa, N., Miyoshi, M., Sano, Y., Satoh, H., Hirano, H., Sakai, H., Nagato, Y., 2003. SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice. Development 130, 705-718.

Pacak, A., Geisler, K., Jorgensen, B., Barciszewska-Pacak, M., Nilsson, L., Nielsen, T.H., Johansen, E., Gronlund, M., Jakobsen, I., Albrechtsen, M., 2010. Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat. Plant Methods 6, 26.

Pan, Z.J., Cheng, C.C., Tsai, W.C., Chung, M.C., Chen, W.H., Hu, J.M., Chen, H.H., 2011. The duplicated B-class MADS-box genes display dualistic characters in orchid floral organ identity and growth. Plant and Cell Physiology 52, 1515-1531.

Pelaz, S., Ditta, G.S., Baumann, E., Wisman, E., Yanofsky, M.F., 2000. B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature 405, 200-203.

Pelaz, S., Tapia-Lopez, R., Alvarez-Buylla, E.R., Yanofsky, M.F., 2001. Conversion of leaves into petals in Arabidopsis. Current Biology 11, 182-184.

Pelucchi, N., Fornara, F., Favalli, C., Masiero, S., Lago, C., Pe, M.E., Colombo, L., Kater, M.M., 2002. Comparative analysis of rice MADS-box genes expressed during flower development. Sexual Plant Reproduction 15, 113-122.

Pflieger, S., Blanchet, S., Camborde, L., Drugeon, G., Rousseau, A., Noizet, M., Planchais, S., Jupin, I., 2008. Efficient virus-induced gene silencing in Arabidopsis using a 'one-step' TYMV-derived vector. The Plant Journal 56, 678-690.

Pinyopich, A., Ditta, G.S., Savidge, B., Liljegren, S.J., Baumann, E., Wisman, E., Yanofsky, M.F., 2003. Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature 424, 85-88.

Prasad, K., Sriram, P., Kumar, C.S., Kushalappa, K., Vijayraghavan, U., 2001. Ectopic expression of rice OsMADS1 reveals a role in specifying the lemma and palea, grass floral organs analogous to sepals. Development Genes and Evolution 211, 281-290.

Prasad, K., Vijayraghavan, U., 2003. Double-stranded RNA interference of a rice PI/GLO paralog, OsMADS2, uncovers its second-whorl-specific function in floral organ patterning. Genetics 165, 2301-2305.

Purkayastha, A., Dasgupta, I., 2009. Virus-induced gene silencing: a versatile tool for discovery of gene functions in plants. Plant Physiology and Biochemistry 47, 967-976.

Ratcliff, F., Martin-Hernandez, A.M., Baulcombe, D.C., 2001. Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing. The Plant Journal 25, 237-245.

Reeves, P.H., Coupland, G., 2001. Analysis of flowering time control in Arabidopsis by comparison of double and triple mutants. Plant Physiology 126, 1085-1091.

Renner, T., Bragg, J., Driscoll, H.E., Cho, J., Jackson, A.O., Specht, C.D., 2009. Virus-induced gene silencing in the culinary ginger (Zingiber officinale): an effective mechanism for down-regulating gene expression in tropical monocots. Molecular Plant 2, 1084-1094.

Riechmann, J.L., Meyerowitz, E.M., 1998. The AP2/EREBP family of plant transcription factors. Biological Chemistry 379, 633-646.

Ruiz, M.T., Voinnet, O., Baulcombe, D.C., 1998. Initiation and maintenance of virus-induced gene silencing. The Plant Cell 10, 937-946.

Salemme, M., Sica, M., Gaudio, L., Aceto, S., 2011. Expression pattern of two paralogs of the PI/GLO-like locus during Orchis italica (Orchidaceae, Orchidinae) flower development. Development Genes and Evolution 221, 241-246.
Salemme, M., Sica, M., Gaudio, L., Aceto, S., 2013. The OitaAG and OitaSTK genes of the orchid Orchis italica: a comparative analysis with other C- and D-class MADS-box genes. Molecular Biology Reports 40, 3523-3535.

Sasaki, S., Yamagishi, N., Yoshikawa, N., 2011. Efficient virus-induced gene silencing in apple, pear and Japanese pear using Apple latent spherical virus vectors. Plant Methods 7, 15.

Sawa, M., Nusinow, D.A., Kay, S.A., Imaizumi, T., 2007. FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis. Science 318, 261-265.

Scofield, S.R., Huang, L., Brandt, A.S., Gill, B.S., 2005. Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiology 138, 2165-2173.

Sirisawat, S., Ezura, H., Fukuda, N., Kounosu, T., Handa, T., 2010. Ectopic expression of an AP3-like and a PI-like genes from 'Sekkoku' orchid (Dendrobium moniliforme) causes the homeotic conversion of sepals to petals in whorl 1 and the suppression of carpel development in whorl 4 in Arabidopsis flowers. Plant Biotechnology 27, 183-192.

Sirisawat, S., Fukuda, N., Ezura, H., Handa, T., 2009. DMMADS4, a DEF-like gene from Dendrobium is required for floral organ identity and flower longevity of orchid. Acta Horticulturae 836, 259-264.

Skipper, M., Johansen, L.B., Pedersen, K.B., Frederiksen, S., Johansen, B.B., 2006. Cloning and transcription analysis of an AGAMOUS- and SEEDSTICK ortholog in the orchid Dendrobium thyrsiflorum (Reichb. f.). Gene 366, 266-274.

Skipper, M., Pedersen, K.B., Johansen, L.B., Frederiksen, S., Irish, V.F., Johansen, B.B., 2005. Identification and quantification of expression levels of three FRUITFULL-like MADS-box genes from the orchid Dendrobium thyrsiflorum (Reichb. f.). Plant Science 169, 579-586.

Sommer, H., Beltran, J.P., Huijser, P., Pape, H., Lonnig, W.E., Saedler, H., Schwarz-Sommer, Z., 1990. Deficiens, a homeotic gene involved in the control of flower morphogenesis in Antirrhinum majus: the protein shows homology to transcription factors. EMBO Journal 9, 605-613.

Song, I.J., Nakamura, T., Fukuda, T., Yokoyama, J., Ito, T., Ichikawa, H., Horikawa, Y., Kameya, T., Kanno, A., 2006. Spatiotemporal expression of duplicate AGAMOUS orthologues during floral development in Phalaenopsis. Development Genes and Evolution 216, 301-313.

Song, Y., Gao, Z., Luan, W., 2012. Interaction between temperature and photoperiod in regulation of flowering time in rice. Science China-Life Sciences 55, 241-249.

Tao, X., Zhou, X., 2004. A modified viral satellite DNA that suppresses gene expression in plants. The Plant Journal 38, 850-860.

Theissen, G., Saedler, H., 2001. Plant biology. Floral quartets. Nature 409, 469-471.

Thiruvengadam, M., Chung, I.M., Yang, C.H., 2012. Overexpression of Oncidium MADS box (OMADS1) gene promotes early flowering in transgenic orchid (Oncidium Gower Ramsey). Acta Physiologiae Plantarum 34, 1295-1302.

Tsai, W.C., Kuoh, C.S., Chuang, M.H., Chen, W.H., Chen, H.H., 2004. Four DEF-like MADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid. Plant and Cell Physiology 45, 831-844.

Tsai, W.C., Lee, P.F., Chen, H.I., Hsiao, Y.Y., Wei, W.J., Pan, Z.J., Chuang, M.H., Kuoh, C.S., Chen, W.H., Chen, H.H., 2005. PeMADS6, a GLOBOSA/PISTILLATA-like gene in Phalaenopsis equestris involved in petaloid formation, and correlated with flower longevity and ovary development. Plant and Cell Physiology 46, 1125-1139.

Tsai, W.C., Pan, Z.J., Hsiao, Y.Y., Jeng, M.F., Wu, T.F., Chen, W.H., Chen, H.H., 2008. Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid. Plant and Cell Physiology 49, 814-824.

Turnage, M.A., Muangsan, N., Peele, C.G., Robertson, D., 2002. Geminivirus-based vectors for gene silencing in Arabidopsis. The Plant Journal 30, 107-114.

Unver, T., Budak, H., 2009. Virus-induced gene silencing, a post transcriptional gene silencing method. International Journal of Plant Genomics 2009, 198680 (Epub.).

Wang, S.Y., Lee, P.F., Lee, Y.I., Hsiao, Y.Y., Chen, Y.Y., Pan, Z.J., Liu, Z.J., Tsai, W.C., 2011. Duplicated C-class MADS-box genes reveal distinct roles in gynostemium development in Cymbidium ensifolium (Orchidaceae). Plant and Cell Physiology 52, 563-577.

Wang, Y.M., Yan, D.W., Y.Y., Z., Li, J., Chang, J., 2012. Regulation of floral organ identity in Arabidopsis by ectopic expression of OsMADS58. Journal of Northeast Agricultural University 19, 60-66.

Wege, S., Scholz, A., Gleissberg, S., Becker, A., 2007. Highly efficient virus-induced gene silencing (VIGS) in California poppy (Eschscholzia californica): an evaluation of VIGS as a strategy to obtain functional data from non-model plants. Annals of Botany 100, 641-649.

Wilson, R.N., Heckman, J.W., Somerville, C.R., 1992. Gibberellin is required for flowering in Arabidopsis thaliana under short days. Plant Physiology 100, 403-408.

Xiang, L., Qin, D.H., Li, X.B., Li, B.J., Guo, F.Q., Wu, C., Sun, C.B., 2011. Cloning and expression analysis of B class MADS-box genes from Cymbidium faberi. Acta Horticulturae Sinica 38, 2333-2341.

Xiao, H., Wang, Y., Liu, D., Wang, W., Li, X., Zhao, X., Xu, J., Zhai, W., Zhu, L., 2003. Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference. Plant Molecular Biology 52, 957-966.

Xu, G., Kong, H., 2007. Duplication and divergence of floral MADS‐Box genes in grasses: evidence for the generation and modification of novel regulators. Journal of Integrative Plant Biology 49, 927-939.

Xu, Y., Teo, L.L., Zhou, J., Kumar, P.P., Yu, H., 2006. Floral organ identity genes in the orchid Dendrobium crumenatum. The Plant Journal 46, 54-68.

Xu, Y., Yu, H., Kumar, P.P., 2010. Characterization of floral organ identity genes of the orchid Dendrobium crumenatum. Asia-Pacific Journal of Molecular Biology and Biotechnology 18, 185-187.

Yadav, S.R., Prasad, K., Vijayraghavan, U., 2007. Divergent regulatory OsMADS2 functions control size, shape and differentiation of the highly derived rice floret second-whorl organ. Genetics 176, 283-294.

Yamaguchi, T., Lee, D.Y., Miyao, A., Hirochika, H., An, G., Hirano, H.-Y., 2006. Functional diversification of the two C-class MADS box genes OSMADS3 and OSMADS58 in Oryza sativa. The Plant Cell 18, 15-28.

Yanofsky, M.F., Ma, H., Bowman, J.L., Drews, G.N., Feldmann, K.A., Meyerowitz, E.M., 1990. The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346, 35-39.

Yanovsky, M.J., Kay, S.A., 2002. Molecular basis of seasonal time measurement in Arabidopsis. Nature 419, 308-312.

Ye, J., Qu, J., Bui, H.T., Chua, N.H., 2009. Rapid analysis of Jatropha curcas gene functions by virus-induced gene silencing. Plant Biotechnology Journal 7, 964-976.

Yoshida, H., Itoh, J., Ohmori, S., Miyoshi, K., Horigome, A., Uchida, E., Kimizu, M., Matsumura, Y., Kusaba, M., Satoh, H., Nagato, Y., 2007. superwoman1-cleistogamy, a hopeful allele for gene containment in GM rice. Plant Biotechnology Journal 5, 835-846.

Yoshida, H., Nagato, Y., 2011. Flower development in rice. Journal of Experimental Botany 62, 4719-4730.

Yu, H., Goh, C.J., 2000. Identification and characterization of three orchid MADS-box genes of the AP1/AGL9 subfamily during floral transition. Plant Physiology 123, 1325-1336.

Yu, H., Yang, S.H., Goh, C.J., 2000. DOH1, a class 1 knox gene, is required for maintenance of the basic plant architecture and floral transition in orchid. The Plant Cell 12, 2143-2160.

Yu, H., Yang, S.H., Goh, C.J., 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.

Yun, D., Liang, W., Dreni, L., Yin, C., Zhou, Z., Kater, M.M., Zhang, D., 2013. OsMADS16 genetically interacts with OsMADS3 and OsMADS58 in specifying floral patterning in rice. Molecular Plant 6, 743-756.

Zahn, L.M., Kong, H., Leebens-Mack, J.H., Kim, S., Soltis, P.S., Landherr, L.L., Soltis, D.E., Depamphilis, C.W., Ma, H., 2005. The evolution of the SEPALLATA subfamily of MADS-box genes: a preangiosperm origin with multiple duplications throughout angiosperm history. Genetics 169, 2209-2223.

Zhang, C., Bradshaw, J.D., Whitham, S.A., Hill, J.H., 2010. The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing. Plant Physiology 153, 52-65.

Zhang, C., Ghabrial, S.A., 2006. Development of bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean. Virology 344, 401-411.

Zheng, S.J., Snoeren, T.A., Hogewoning, S.W., van Loon, J.J., Dicke, M., 2010. Disruption of plant carotenoid biosynthesis through virus-induced gene silencing affects oviposition behaviour of the butterfly Pieris rapae. New Phytologist 186, 733-745.