Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

A protocol for Agrobacterium-mediated transformation in rice


Agrobacterium-mediated transformation of rice is an important method that has been widely adopted by many laboratories. However, because current approaches rely on culture systems, routine protocols have been established only in japonica rice, especially those varieties with higher regeneration potential. Some very efficient methods have been developed for japonica varieties that enable high-throughput functional analysis in rice; however, many elite japonica, and most indica, varieties are difficult to regenerate, leading to low transformation efficiencies. Much effort has been devoted to improving transformation efficiency for all rice genotypes. Here, we describe an Agrobacterium-mediated rice transformation method that is applicable to easily cultured varieties in addition to elite japonica varieties that are more difficult to culture. Using this method, transgenic rice plants can be obtained in about 2–3 months with a transformation frequency of 30–50%, both in easily cultured varieties and recalcitrant elite japonica rice.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 2: Procedure for producing transgenic rice plants.
Figure 1: Regeneration test of three rice varieties.

Similar content being viewed by others


  1. Hiei, Y., Ohta, S., Komari, T. & Kumashiro, T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271–282 (1994).

    Article  CAS  Google Scholar 

  2. Hiei, Y., Komari, T. & Kubo, T. Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol. Biol. 35, 205–218 (1997).

    Article  CAS  Google Scholar 

  3. Toki, S. Rapid and efficient Agrobacterium-mediated transformation in rice. Plant Mol. Biol. Rep. 15, 16–21 (1997).

    Article  CAS  Google Scholar 

  4. Cho, S.K. et al. Efficient transformation of korean rice cultivars (Oryza sativa L.) mediated by Agrobacterium tumefaciens. J. Plant Biol. 41, 262–268 (1998).

    Article  Google Scholar 

  5. Yara, A. et al. Production of transgenic japonica rice (Oryza sativa) cultivar, Taichung 65, by the Agrobacterium-mediated method. Plant Biotech. 18, 305–310 (2001).

    Article  CAS  Google Scholar 

  6. Rashid, H., Yokoi, S., Toriyama, K. & Hinata, K. Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Rep. 15, 727–730 (1996).

    Article  CAS  Google Scholar 

  7. Aldemita, R.R. & Hodges, T.K. Agrobacterium tumefaciens-mediated tansformation of japonica and indica rice varieties. Planta 199, 612–617 (1996).

    Article  CAS  Google Scholar 

  8. Zhang, J., Xu, R., Elliott, M.C. & Chen, D.-F. Agrobacterium-mediated transformation of elite indica and japonica rice cultivars. Mol. Biotech. 8, 223–231 (1997).

    Article  CAS  Google Scholar 

  9. Dong, J., Teng, W., Buchholz, W.G. & Hall, T.C. Agrobacterium-mediated transformation of javanica rice. Mol. Breed. 2, 267–276 (1996).

    Article  CAS  Google Scholar 

  10. Rachmawati, D., Hosaka, T., Inoue, E. & Anzai, H. Agrobacterium-mediated transformation of Javanica rice cv. Rojolele. Biosci. Biotechnol. Biochem., 68, 1193–1200 (2004).

    Article  CAS  Google Scholar 

  11. Jeon, J.S. et al. T-DNA insertion mutagenesis for functional genomics in rice. Plant J. 22, 561–570 (2000).

    Article  CAS  Google Scholar 

  12. Jeong, D.H. et al. T-DNA insertional mutagenesis for activation tagging in rice. Plant Physiol. 130, 1636–1644 (2002).

    Article  CAS  Google Scholar 

  13. Wu, C.Y. et al. Development of enhancer trap lines for functional analysis of the rice genome. Plant J. 35, 418–427 (2003).

    Article  CAS  Google Scholar 

  14. Sha, Y. et al. Generation and flanking sequence analysis of a rice T-DNA tagged population. Theor. Appl. Genet., 108, 306–314 (2004).

    Article  CAS  Google Scholar 

  15. Sallaud, C. et al. Highly efficient production and characterization of T-DNA plants for trice (Oryza sativa L.) functional genomics. Theor. Appl. Genot. 106, 1396–1408 (2003).

    Article  CAS  Google Scholar 

  16. Murashige, T. & Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497 (1962).

    Article  CAS  Google Scholar 

  17. Chu, C.-C., Wang, C.-C. & Sun, C.-S. Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci. Sin. 18, 659–668 (1975).

    Google Scholar 

  18. Nishimura, A. et al. Isolation of a rice regeneration QTL gene and its application to new transformation system. Proc. Natl. Acad. Sci. USA 102, 11940–11944 (2005).

    Article  CAS  Google Scholar 

  19. Hoa, T.T.C. et al. Golden indica and japonica rice lines amenable to deregulation. Plant Physiol. 133, 161–169 (2003).

    Article  CAS  Google Scholar 

  20. Kumar, K.K. et al. An improved Agrobacterium-mediated transformation protocol for recalcitrant elite indica rice cultivars. Plant Mol. Biol. Rep. 23, 67–73 (2005).

    Article  CAS  Google Scholar 

  21. Ignacimuthu, S. & Arockiasamy, S. Agrobacterium-mediated transformation of an elite indica rice for insect resistance. Curr. Sci. 90, 829–835 (2006).

    CAS  Google Scholar 

  22. Supartana, P. et al. Development of simple and efficient in planta transformation method for rice (Oryza sativa L.) using Agrobacterium tumefaciens. J. Biosci. Bioeng. 100, 391–397 (2005).

    Article  CAS  Google Scholar 

  23. Sentoku, N., Sato, Y. & Matsuoka, M. Overexpression of rice OSH genes induces ectopic shoots on leaf sheaths of transgenic rice plants. Dev. Biol. 220, 358–364 (2000).

    Article  CAS  Google Scholar 

  24. Nishimura, A. et al. OsPNH1 regulates leaf development and maintenance of the shoot apical meristem in rice. Plant J. 30, 189–201 (2002).

    Article  CAS  Google Scholar 

  25. Ashikari, M. et al. Cytokinin oxidase regulates rice grain production. Science 309, 741–745 (2005).

    Article  CAS  Google Scholar 

  26. Hood, E.E., Helmer, G.L., Fraley, R.T. & Chilton, M.D. The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168, 1291–1301 (1986).

    Article  CAS  Google Scholar 

  27. Hood, E.E., Gelvin, S.B., Melches, L.S. & Hoekema, A. New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2, 208–218 (1993).

    Article  CAS  Google Scholar 

  28. Ohta, S., Mita, S., Hattori, T. & Nakamura, K. Construction and expression in tobacco of a β glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol. 31, 805–813 (1990).

    CAS  Google Scholar 

  29. Zhang, W., McElroy, D. & Wu, R. Analysis of rice Act1 5′ region activity in transgenic rice plants. Plant Cell 3, 1155–1165 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Joy, K.W. & Hageman, R.H. The purification and properties of nitrite reductase from higher plants, and its dependence on ferredoxin. Biochem. J. 100, 263–273 (1966).

    Article  CAS  Google Scholar 

  31. Sanderson, G.W. & Cocking, E.C. Enzymic assimilation of nitrate in tomato plants. I. Reduction of nitrate to nitrite. Plant Physiol. 39, 416–422 (1964).

    Article  CAS  Google Scholar 

  32. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    Article  CAS  Google Scholar 

  33. Misko, T.P. et al. A fluorometric assay for the measurement of nitrite in biological samples. Anal. Biochem. 214, 11–16 (1993).

    Article  CAS  Google Scholar 

Download references


We thank Sakimi Watanabe and Sachiko Nomura for their technical assistance in establishing this protocol.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Asuka Nishimura.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nishimura, A., Aichi, I. & Matsuoka, M. A protocol for Agrobacterium-mediated transformation in rice. Nat Protoc 1, 2796–2802 (2006).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing