Plant Cell Reports

, Volume 25, Issue 12, pp 1355–1361 | Cite as

A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean

  • Keito Nishizawa
  • Yoichi Kita
  • Masahiko Kitayama
  • Masao Ishimoto
Genetic Transformation and Hybridization


Fluorescent proteins such as green fluorescent protein (GFP) from Aequorea victoria are often used as markers for transient expression and stable transformation in plants, given that their detection does not require a substrate and they can be monitored in a nondestructive manner. We have now evaluated the red fluorescent protein DsRed2 (a mutant form of DsRed from Discosoma sp.) for its suitability as a visual marker in combination with antibiotic selection for genetic transformation of soybean [Glycine max (L.) Merrill]. Transient and stable expression of DsRed2 in somatic embryos was readily detected by fluorescence microscopy, allowing easy confirmation of gene introduction. We obtained several fertile transgenic lines, including homozygous lines, that grew and produced seeds in an apparently normal manner. The red fluorescence of DsRed2 was detected by fluorescence microscopy without background fluorescence in both leaves and seeds of the transgenic plants. Furthermore, in contrast to seeds expressing GFP, those expressing DsRed2 were readily identifiable even under white light by the color conferred by the transgene product. The protein composition of seeds was not affected by the introduction of DsRed2, with the exception of the accumulation of DsRed2 itself, which was detectable as an additional band on electrophoresis. These results indicate that DsRed2 is a suitable reporter (even more suitable than GFP) for genetic transformation of soybean.


Bombardment DsRed2 Fluorescent protein Nondestructive Transgenic soybean 



This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). We thank Y. Niwa for providing the sGFP(S65T) reporter gene.


  1. Clontech (2001) Living Colors™ DsRed2, Improved red fluorescent protein for use in living cells. Clontechniques
  2. Dietrich C, Maiss E (2002) Red fluorescent protein DsRed from Discosoma sp. as a reporter protein in higher plants. Biotechniques 32:286–293PubMedGoogle Scholar
  3. El-Shemy HA, Teraishi M, Khalafalla MM, Katsube-Tanaka T, Utsumi S, Ishimoto M (2004) Isolation of soybean plants with stable transgene expression by visual selection based on green fluorescent protein. Mol Breed 14:227–238CrossRefGoogle Scholar
  4. Finer KR, Finer JJ (2000) Use of Agrobacterium expressing green fluorescent protein to evaluate colonization of sonication-assisted Agrobacterium-mediated transformation-treated soybean cotyledons. Lett Appl Microbiol 30:406–410PubMedCrossRefGoogle Scholar
  5. Furutani N, Hidaka S (2004) Efficient production of transgenic soybean using a co-transformation method. Breed Sci 54:91–98CrossRefGoogle Scholar
  6. Gilson PR, Vergara CE, Kjer-Nielsen L, Teasdale RD, Bacic A, Gleeson PA (2004) Identification of a Golgi-localised GRIP domain protein from Arabidopsis thaliana. Planta 219:1050–1056PubMedCrossRefGoogle Scholar
  7. Goodin MM, Dietzgen RG, Schichnes D, Ruzin S, Jackson AO (2002) pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. Plant J 31:375–383PubMedCrossRefGoogle Scholar
  8. Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA 94:2122–2127PubMedCrossRefGoogle Scholar
  9. Jach G, Binot E, Frings S, Luxa K, Schell J (2001) Use of red fluorescent protein from Discosoma sp. (dsRED) as a reporter for plant gene expression. Plant J 28:483–491PubMedCrossRefGoogle Scholar
  10. Khalafalla MM, Rhaman SM, El-Shemy HA, Nakamoto Y, Wakasa K, Ishimoto M (2005) Optimization of particle bombardment conditions by monitoring of transient sGFP(S65T) expression in transformed soybean. Breed Sci 55:257–263CrossRefGoogle Scholar
  11. Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17:969–973PubMedCrossRefGoogle Scholar
  12. Matzke AJ, Huettel B, Van Der Winden J, Matzke M (2005) Use of two-color fluorescence-tagged transgenes to study interphase chromosomes in living plants. Plant Physiol 139:1586–1596PubMedCrossRefGoogle Scholar
  13. Ponappa T, Brzozowski AE, Finer JJ (1999) Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein. Plant Cell Rep 19:6–12CrossRefGoogle Scholar
  14. Reichel C, Mathur J, Eckes P, Langenkemper K, Koncz C, Schell J, Reiss B, Maas C (1996) Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells. Proc Natl Acad Sci USA 93:5888–5893PubMedCrossRefGoogle Scholar
  15. Stewart Jr CN (2001) The utility of green fluorescent protein in transgenic plants. Plant Cell Rep 20:376–382CrossRefGoogle Scholar
  16. Stewart Jr CN (2005) Monitoring the presence and expression of transgenes in living plants. Trends Plant Sci 10:390–396CrossRefGoogle Scholar
  17. Wenck A, Pugieux C, Turner M, Dunn M, Stacy C, Tiozzo A, Dunder E, van Grinsven E, Khan R, Sigareva M, Wang WC, Reed J, Drayton P, Oliver D, Trafford H, Legris G, Rushton H, Tayab S, Launis K, Chang YF, Chen DF, Melchers L (2003) Reef-coral proteins as visual, non-destructive reporters for plant transformation. Plant Cell Rep 22:244–251PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Keito Nishizawa
    • 1
  • Yoichi Kita
    • 2
  • Masahiko Kitayama
    • 2
  • Masao Ishimoto
    • 1
  1. 1.National Agricultural Research Center for Hokkaido RegionHokkaidoJapan
  2. 2.Ehime Women’s CollegeEhimeJapan

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