Efficient and Fast Production of Transgenic Rice Plants by Agrobacterium-Mediated Transformation

  • Chuanyin WuEmail author
  • Yi Sui
Part of the Methods in Molecular Biology book series (MIMB, volume 1864)


Genetic transformation plays a key role in deciphering regulation of agronomic traits at molecular level in rice, a model monocot cereal crop. Here we describe an efficient and fast protocol for producing transgenic japonica rice plants using the Agrobacterium-mediated transformation method. The protocol simplifies medium compositions and transformation steps and can be easily followed by a lab technician with little tissue culture experience. Using this protocol, we have transformed thousands of gene constructs in the past 10 years and edited hundreds of genes with the CRISPR-Cas9 system recently.

Key words

Rice Agrobacterium-mediated transformation Callus induction Selection of transformants Plant regeneration 



This work was supported by the Innovation Program of Chinese Academy of Agricultural Sciences.


  1. 1.
    Shimamoto K, Terada R, Izawa T, Fujimoto H (1989) Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338:274–276CrossRefGoogle Scholar
  2. 2.
    Christou P, Ford T, Kofron M (1991) Production of transgenic rice (Oryza Sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Nat Biotechnol 9:957–962CrossRefGoogle Scholar
  3. 3.
    Dai S, Zheng P, Marmey P, Zhang S, Tian W, Chen S, Beachy RN, Fauquet C (2001) Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed 7:25–33CrossRefGoogle Scholar
  4. 4.
    Chen L, Marmey P, Taylor NJ, Brizard JP, Espinoza C, D'Cruz P, Huet H, Zhang S, Kochko A, Beachy RN, Fauquet CM (1998) Expression and inheritance of multiple transgenes in rice plants. Nat Biotechnol 16:1060–1064CrossRefGoogle Scholar
  5. 5.
    Zhu C, Naqvi S, Breitenbach G, Sandmann J, Christou P, Capell T (2008) Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize. Proc Natl Acad Sci U S A 105:18232–18237CrossRefGoogle Scholar
  6. 6.
    Naqvi S, Zhu C, Farre G, Bassie L, Ramessar K, Breitenbach J, Perez-Conesa D, Ros-Berruezo G, Sandmann G, Capell T, Christou P (2009) Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc Natl Acad Sci U S A 106:7762–7767CrossRefGoogle Scholar
  7. 7.
    Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, Qiu JL, Gao C (2016) Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun 7:12617. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Svitashev S, Schwartz C, Lenderts B, Young JK, Cigan AK (2016) Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat Commun 7:13274. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu C, Ran Y, Gao C (2017) Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun 8:14261. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Rained DM, Bottino P, Gordon MP, Nester EW (1990) Agrobecterium-mediated transformation of rice (Oryza sativa L.). Nat Biotechnol 8:33–38CrossRefGoogle Scholar
  11. 11.
    Chan MT, Chang HH, Ho SL, Tong WF, Yu SM (1993) Agrobacteriummediated production of transgenic rice plants expressing a chimeric ɑ-amylase promoter/β glucuronidase gene. Plant Mol Biol 22:491–506CrossRefGoogle Scholar
  12. 12.
    Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282CrossRefGoogle Scholar
  13. 13.
    Hiei Y, Komari T (2008) Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc 3:824–834CrossRefGoogle Scholar
  14. 14.
    Roy M, Jain RK, Rohila JS, Wu R (2000) Production of agronomically superior transgenic rice plants using Agrobacterium transformation methods: present status and future perspectives. Curr Sci 79:954–960Google Scholar
  15. 15.
    Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka H (2006) Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J 47:969–976CrossRefGoogle Scholar
  16. 16.
    Zhou F, Lin Q, Zhu L et al (2013) D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature 504:406–410CrossRefGoogle Scholar
  17. 17.
    Gao H, Jin M, Zheng X et al (2014) Days to heading 7, a major quantitative locus determining photoperiod sensitivity and regional adaptation in rice. Proc Natl Acad Sci U S A 46:16337–16342CrossRefGoogle Scholar
  18. 18.
    Liu Y, Wu H, Chen H et al (2015) A gene cluster encoding lectin receptor kinases confers broad-spectrum and durable insect resistance in rice. Nat Biotechnol 33:301–305CrossRefGoogle Scholar
  19. 19.
    Wu S, Xie Y, Zhang J et al (2015) VLN2 regulates plant architecture by affecting microfilament dynamics and polar auxin transport in rice. Plant Cell 27:2829–2845CrossRefGoogle Scholar
  20. 20.
    Sun Y, Zhang X, Wu C, He Y, Ma Y, Hou H, Guo X, Du W, Zhao Y, Xia L (2016) Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase. Mol Plant 9:628–631CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.The National Key Facility for Crop Gene Resources and Genetic ImprovementInstitute of Crop Sciences, Chinese Academy of Agricultural SciencesBeijingChina

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