Molecular Breeding

, Volume 30, Issue 2, pp 671–680

Flower color alteration in the liliaceous ornamental Tricyrtis sp. by RNA interference-mediated suppression of the chalcone synthase gene

  • Yukiko Kamiishi
  • Masahiro Otani
  • Hiroki Takagi
  • Dong-Sheng Han
  • Shiro Mori
  • Fumi Tatsuzawa
  • Hiroaki Okuhara
  • Hitoshi Kobayashi
  • Masaru Nakano


Chalcone synthase (CHS) is the key enzyme in an early stage of the flavonoid biosynthetic pathway. In the present study, a full-length cDNA clone for CHS was isolated from flower tepals of the liliaceous ornamental Tricyrtis sp., in which tepals have many reddish-purple spots resulting from accumulation of cyanidin derivatives. The deduced amino acid sequence of the isolated cDNA clone, designated TrCHS1 (accession number AB478624 in the GenBank/EMBL/DDBJ databases), shows 79.4–91.4% identity with those of previously reported CHS genes. An RNA interference (RNAi) construct targeting TrCHS1 was introduced by Agrobacterium-mediated transformation in order to alter the flower color of Tricyrtis sp. Seven transgenic plants that produced flowers could be classified into three types according to flower color phenotype: one transgenic plant had tepals with as many reddish-purple spots as non-transgenic plants (Type I); one had tepals with reduced numbers of reddish-purple spots (Type II); and five had completely white tepals without any spots (Type III). High-performance liquid chromatography analysis showed that tepals of Type III transgenic plants did not accumulate detectable amounts of anthocyanidins. In addition, TrCHS1 mRNA levels in tepals of Type II and Type III transgenic plants decreased substantially compared with non-transgenic plants, as determined by quantitative real-time reverse transcription–polymerase chain reaction analysis. Our results indicate the validity of RNAi suppression of the flavonoid biosynthetic pathway genes for flower color alteration in Tricyrtis sp. To the best of our knowledge, this is the first report on flower color alteration by genetic transformation in monocotyledonous ornamentals.


Agrobacterium-mediated transformation Anthocyanidin Monocotyledonous ornamental RNAi Transgenic plant 

Supplementary material

11032_2011_9653_MOESM1_ESM.docx (224 kb)
Supplementary material 1 (DOCX 224 kb)


  1. Adachi Y, Mori S, Nakano M (2005) Agrobacterium-mediated production of transgenic plants in Tricyrtis hirta (Liliaceae). Acta Hort 673:415–419Google Scholar
  2. Chuang CF, Meyerowitz EM (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA 97:4985–4990PubMedCrossRefGoogle Scholar
  3. Courtney-Gutterson N, Napoli C, Lemieux C, Morgan A, Firoozabady E, Robinson KE (1994) Modification of flower color in florist’s chrysanthemum: production of a white-flowering variety through molecular genetics. Biotechnology 12:268–271PubMedCrossRefGoogle Scholar
  4. de Mesa MC, Santiago-Doménech N, Pliego-Alfaro F, Quesada MA, Mercado JA (2004) The CaMV 35S promoter is highly active on floral organs and pollen of transgenic strawberry plants. Plant Cell Rep 23:32–38PubMedCrossRefGoogle Scholar
  5. Deroles SC, Bradley JM, Schwinn KE, Markham KR, Bloor S, Manson DG, Davies KM (1998) An antisense chalcone synthase cDNA leads to novel colour patterns in lisianthus (Eustoma grandiflorum) flowers. Mol Breed 4:59–66CrossRefGoogle Scholar
  6. Durbin ML, Learn GH Jr, Huttley GA, Clegg MT (1995) Evolution of the chalcone synthase gene family in the genus Ipomoea. Proc Natl Acad Sci USA 92:3338–3342PubMedCrossRefGoogle Scholar
  7. Elomaa P, Honkanen J, Puska R, Seppäne P, Helariutta Y, Mehto M, Kotilainen M, Nevalainen L, Teeri TH (1993) Agrobacterium-mediated transfer of antisense chalcone synthase cDNA to Gerbera hybrida inhibits flower pigmentation. Biotechnology 11:508–511CrossRefGoogle Scholar
  8. Forkmann G (1991) Flavonoids as flower pigments: the formation of the natural spectrum and its extension by genetic engineering. Plant Breed 106:1–26CrossRefGoogle Scholar
  9. Gutterson N (1995) Anthocyanin biosynthetic genes and their application to flower color modification through sense suppression. HortSci 30:964–966Google Scholar
  10. Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071–1083PubMedGoogle Scholar
  11. Hoshi Y, Kondo M, Mori S, Adachi Y, Nakano M, Kobayashi H (2004) Production of transgenic lily plants by Agrobacterium-mediated transformation. Plant Cell Rep 22:359–364PubMedCrossRefGoogle Scholar
  12. Johzuka-Hisatomi Y, Hoshino A, Mori T, Habu Y, Iida S (1999) Characterization of the chalcone synthase genes expressed in flowers of the common and Japanese morning glories. Genet Syst 74:141–147CrossRefGoogle Scholar
  13. Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195PubMedCrossRefGoogle Scholar
  14. Katsumoto Y, Fukushi-Mizutani M, Fukui Y, Brugliera F, Holton TA, Karan M, Nakamura N, Yonekura-sakakibara K, Togami J, Pigeaire A, Tao G, Nehra N, Lu C, Dyson B, Tsuda S, Ashikari T, Kusumi T, Mason J, Tanaka Y (2007) Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. Plant Cell Physiol 48:1589–1600PubMedCrossRefGoogle Scholar
  15. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120PubMedCrossRefGoogle Scholar
  16. Koes RE, Spelt CE, van den Elzen PJM, Mol JNM (1989) Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida. Gene 81:245–257PubMedCrossRefGoogle Scholar
  17. Li J, Ou-Lee T-M, Raba R, Amundson RG, Last RL (1993) Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant Cell 5:171–179PubMedGoogle Scholar
  18. Mattanovich D, Rüker F, Machado AC, Laimer M, Regner F, Steinkellner H, Himmler G, Katinger H (1989) Efficient transformation of Agrobacterium spp. by electroporation. Nucleic Acids Res 17:6747PubMedCrossRefGoogle Scholar
  19. Meyer P, Heidmann I, Forkmann G, Saedler H (1987) A new petunia flower colour generated by transformation of a mutant with a maize gene. Nature 330:667–678CrossRefGoogle Scholar
  20. Miki D, Shimamoto K (2004) Simple RNAi vectors for stable and transient suppression of gene function in rice. Plant Cell Physiol 45:490–495PubMedCrossRefGoogle Scholar
  21. Miki D, Itoh R, Shimamoto K (2005) RNA silencing of single and multiple members in a gene family of rice. Plant Physiol 138:1903–1913PubMedCrossRefGoogle Scholar
  22. Mori S, Asano S, Kobayashi H, Nakano M (2002) Analysis of anthocyanidins and anthocyanins in flowers of Muscari spp. Bull Fac Agric Niigata Univ 55:13–18Google Scholar
  23. Mori S, Oka E, Umehara H, Suzuki S, Kobayashi H, Hoshi Y, Kondo M, Koike Y, Nakano M (2007) Somaclonal variation and stability of GUS gene expression in transgenic agapanthus (Agapanthus praecox ssp. orientalis) plants at the flowering stage. In Vitro Cell Dev Biol Plant 43:79–87CrossRefGoogle Scholar
  24. Mori S, Oka E, Umehara H, Kobayashi H, Hoshi Y, Kondo M, Koike Y, Nakano M (2008) Stability of β-glucuronidase gene expression in transgenic Tricyrtis hirta plants after two years of cultivation. Biol Plant 52:513–516CrossRefGoogle Scholar
  25. Nakamura N, Fukuchi-Mizutani M, Miyazaki K, Suzuki K, Tanaka Y (2006) RNAi suppression of the anthocyanidin synthase gene in Torenia hybrida yields white flowers with higher frequency and better stability than antisense and sense suppression. Plant Biotechnol 23:13–17CrossRefGoogle Scholar
  26. Nakano M, Mizunashi K, Tanaka S, Godo T, Nakata M, Saito H (2004) Somatic embryogenesis and plant regeneration from callus cultures of several species in the genus Tricyrtis. In Vitro Cell Dev Biol Plant 40:274–278CrossRefGoogle Scholar
  27. Nakano M, Mori S, Suzuki S, Hoshi Y, Kobayashi H (2006) Production of transgenic plants via Agrobacterium-mediated transformation in Liliaceous ornamentals. In: da Silva JAT (ed) Floriculture, ornamental and plant biotechnology: advances and topical issues, vol II. Global Science Books, Middlesex, pp 172–183Google Scholar
  28. Nakatsuka T, Izumi Y, Yamagishi M (2003) Spatial and temporal expression of chalcone synthase and dihydroflavonol 4-reductase genes in the Asiatic hybrid lily. Plant Sci 165:759–767CrossRefGoogle Scholar
  29. Nakatsuka T, Mishiba K, Abe Y, Kakizaki Y, Yamamura S, Nishihara M (2008) Flower color modification of gentian plants by RNAi-mediated gene silencing. Plant Biotechnol 25:61–68CrossRefGoogle Scholar
  30. Nishihara M, Nakatsuka T, Hosokawa K, Yokoi T, Abe Y, Mishiba K, Yamamura S (2006) Dominant inheritance of white-flowered and herbicide-resistant traits in transgenic gentian plants. Plant Biotechnol 23:25–31CrossRefGoogle Scholar
  31. Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. CABIOS 12:357–358PubMedGoogle Scholar
  32. Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160PubMedCrossRefGoogle Scholar
  33. Suzuki K, Xue H, Tanaka Y, Fukui Y, Fukuchi-Mizutani M, Murakami Y, Katsumoto Y, Tsuda S, Kusumi T (2000) Flower color modification of Torenia hybrida by cosuppression of anthocyanin biosynthesis genes. Mol Breed 6:239–246CrossRefGoogle Scholar
  34. Tatsuzawa F, Saito N, Miyoshi K, Shinoda K, Shigihara A, Honda T (2004) Diacylated 8-C-glucosylcyanidin 3-glucoside from the flowers of Tricyrtis formosana. Chem Pharm Bull 52:631–633PubMedCrossRefGoogle Scholar
  35. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  36. Tsuda S, Fukui Y, Nakamura N, Katsumoto Y, Yonekura-Sakakibara K, Fukuchi-Mizutani M, Ohira K, Ueyama Y, Ohkawa H, Holton TA, Kusumi T, Tanaka Y (2004) Flower color modification of Petunia hybrida commercial varieties by metabolic engineering. Plant Biotechnol 21:377–386CrossRefGoogle Scholar
  37. Waterhouse PM, Graham MW, Wang M-B (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 95:13959–13964PubMedCrossRefGoogle Scholar
  38. Winkel-Shirley B (2002) Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol 5:218–223PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Yukiko Kamiishi
    • 1
  • Masahiro Otani
    • 1
  • Hiroki Takagi
    • 1
  • Dong-Sheng Han
    • 1
  • Shiro Mori
    • 2
  • Fumi Tatsuzawa
    • 3
  • Hiroaki Okuhara
    • 4
  • Hitoshi Kobayashi
    • 4
  • Masaru Nakano
    • 1
  1. 1.Faculty of Agriculture, Niigata UniversityNiigataJapan
  2. 2.Department of Agro-Environmental ScienceTakushoku University, Hokkaido Junior CollegeFukagawaJapan
  3. 3.Faculty of Agriculture, Iwate UniversityMoriokaJapan
  4. 4.Niigata Agricultural Research InstituteNagaokaJapan

Personalised recommendations