Plant Cell Reports

, Volume 31, Issue 11, pp 2075–2084

Agrobacterium-mediated co-transformation of rice using two selectable marker genes derived from rice genome components

Original Paper


A method for Agrobacterium-mediated co-transformation of rice (Oryza sativa L.) was developed using rice-derived selection markers. Two T-DNAs were efficiently introduced into separate loci using selectable marker gene cassettes consisting of the mutated acetolactate synthase gene (mALS) under the control of the callus-specific promoter (CSP) (CSP:mALS) and the ferredoxin nitrite reductase gene (NiR) under the control of its own promoter (NiR P:NiR). The CSP:mALS gene cassette confers sulfonylurea herbicide resistance to transgenic rice callus. The NiR P:NiR construct complements NiR-deficient mutant cultivars such as ‘Koshihikari’, which are defective in the regulation of nitrogen metabolism. In the present study, the CaMV35S:GUS and CaMV35S:GFP gene cassettes were co-introduced into the ‘Koshihikari’ genome using our system. Approximately 5–10 independent transgenic lines expressing both the GUS and GFP reporters were obtained from 100 Agrobacterium co-inoculated calli. Furthermore, transgenic ‘Koshihikari’ rice lines with reduced content of two major seed allergen proteins, the 33 and 14–16 kDa allergens, were generated by this co-transformation system. The present results indicate that the generation of selectable antibiotic resistance marker gene-free transgenic rice is possible using our rice-derived selection marker co-transformation system.

Key message An improved rice transformation method was developed based on Agrobacterium-mediated co-transformation using two rice genome-derived selectable marker gene cassettes.


Acetolactate synthase Agrobacterium Co-transformation Ferredoxin nitrite reductase GM crop Transgenic rice 



Callus-specific promoter


2,4-Dichlorophenoxyacetic acid


Green fluorescent protein

GM crop

Genetically modified crop




Hygromycin phosphotransferase


Mutated acetolactate synthase


Multi-cloning site


Naphthaleneacetic acid


Ferredoxin nitrite reductase


Neomycin phosphotransferase


  1. Baisakh N, Rehana S, Rai M, Oliva N, Tan J, Maclill D, Khush GS, Datta K, Datta SK (2006) Marker-free transgenic (MFT) near-isogenic introgression lines (NILs) of ‘golden’ indica rice (cv IR64) with accumulation of provitamin A in the endosperm tissue. Plant Biotechnol J 4:467–475PubMedCrossRefGoogle Scholar
  2. Chaleff RW, Mauvais CJ (1984) Acetolactate synthase is the site of action of two sulfonylurea herbicides in higher plants. Science 224:1443–1445PubMedCrossRefGoogle Scholar
  3. Depicker A, Herman L, Jacobs A, Schell J, Van Montague M (1985) Frequency of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Mol Gen Genet 201:477–484CrossRefGoogle Scholar
  4. Ebinuma H, Sugita K, Matsunaga E, Yamakado M (1997) Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc Natl Acad Sci USA 94:2117–2121PubMedCrossRefGoogle Scholar
  5. Endo S, Sugita K, Sakai M, Tanaka H, Ebinuma H (2002) Single-step transformation for generating marker-free transgenic rice using the ipt-type MAT vector system. Plant J 30:115–122PubMedCrossRefGoogle Scholar
  6. Ito S, Rosegrant MW, Agcaoili-Sombilla MC (1995) Quality-equivalent and cost-adjusted measurement of international competitiveness in Japanese rice markets. International Food Policy Research Institute, EPTD discussion paper no. 91, pp 1–23Google Scholar
  7. James C (2011) Global status of commercialized biotech/GM crops: 2011. ISAAA Brief No. 43, ISAAA, Ithaca, NYGoogle Scholar
  8. Kawakatsu T, Hirose S, Yasuda H, Takaiwa F (2010) Reducing rice seed storage protein accumulation leads to changes in nutrient quality and storage organelle formation. Plant Physiol 154:1842–1854PubMedCrossRefGoogle Scholar
  9. Komari T, Hiei Y, Mukai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174PubMedCrossRefGoogle Scholar
  10. Matsuda T, Nakasa M, Alvarez AM, Izumi H, Kato T, Tada Y (2006) Rice-seed allergenic protein and hypoallergenic rice. In: Mine Y, Shahidi F (eds) Nutraceutical proteins and peptides in health and disease. Taylor and Francis, London, pp 493–511Google Scholar
  11. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325PubMedCrossRefGoogle Scholar
  12. Nishimura A, Ashikari M, Lin S, Takashi T, Angeles ER, Yamamoto T, Matsuoka M (2005) Isolation of a rice regeneration quantitative trait loci gene and its application to transformation system. Proc Natl Acad Sci USA 102:11940–11944PubMedCrossRefGoogle Scholar
  13. Okuzaki A, Shimizu T, Kaku K, Kawai K, Toriyama K (2007) A novel mutated acetolactate synthase gene conferring specific resistance to pyrimidinyl carboxy herbicides in rice. Plant Mol Biol 64:219–224PubMedCrossRefGoogle Scholar
  14. Onodera Y, Suzuki A, Wu CY, Washida H, Takaiwa F (2001) A rice functional transcriptional activator, RISBZ1, responsible for endosperm-specific expression of storage protein genes through GCN4 motif. J Biol Chem 276:14139–14152PubMedGoogle Scholar
  15. Ozawa K (2009) Establishment of a high efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.). Plant Sci 176:522–527CrossRefGoogle Scholar
  16. Ozawa K, Kawahigashi H (2006) Positional cloning of the nitrite reductase gene associated with good growth and regeneration ability of calli and establishment of a new selection system for Agrobacterium-mediated transformation in rice (Oryza sativa L.). Plant Sci 170:384–393CrossRefGoogle Scholar
  17. Parkhi V, Ray M, Tan J, Oliva N, Rehana S, Bandyopadhyay A, Torrizo L, Ghole V, Datta K, Datta SK (2005) Molecular characterization of marker-free transgenic lines of indica rice that accumulate carotenoids in seed endosperm. Mol Gen Genomics 274:325–336CrossRefGoogle Scholar
  18. Qu L, Takaiwa F (2004) Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice. Plant Biotechnol J 2:113–125CrossRefGoogle Scholar
  19. Shimizu T, Nakayama I, Nagayama K, Miyazawa T, Nezu Y (2002) Acetolactate synthase inhibitors. In: Boger P, Wakabayashi K, Hirai K (eds) Herbicide classes in development, vol 1. Springer, Berlin, pp 1–41CrossRefGoogle Scholar
  20. Sripriya R, Sangeetha M, Parameswari C, Veluthambi B, Veluthambi K (2011) Improved Agrobacterum-mediated co-transformation and selectable marker elimination in transgenic rice by using a copy number pBIN19-derived binary vector. Plant Sci 180:766–774PubMedCrossRefGoogle Scholar
  21. Sugita K, Kasahara T, Matsunaga E, Ebinuma H (2000) A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency. Plant J 22:461–469PubMedCrossRefGoogle Scholar
  22. Tu J, Datta K, Oliva N, Zhang G, Xu C, Khush GS, Zhang Q, Datta SK (2003) Site-independently integrated transgenes in the elite restorer rice line Minghui 63 allow removal of a selectable marker from the gene of interest by self-segregation. Plant Biotechnol J 1:155–165PubMedCrossRefGoogle Scholar
  23. Wakasa Y, Yasuda H, Takaiwa F (2006) High accumulation of bioactive peptide in transgenic rice seeds by expression of introduced multiple genes. Plant Biotechnol J 4:499–510PubMedGoogle Scholar
  24. Wakasa Y, Ozawa K, Takaiwa F (2007) Agrobacterium-mediated transformation of a low glutelin mutant of ‘Koshihikari’ rice variety using the mutated-acetolactate synthase gene derived from rice genome as a selectable marker. Plant Cell Rep 26:1567–1573PubMedCrossRefGoogle Scholar
  25. Wakasa Y, Hirano K, Uris A, Matsuda M, Takaiwa F (2011) Generation of transgenic rice lines with reduced contents of multiple potential allergens using a null mutant in combination with an RNA silencing method. Plant Cell Physiol 52:2190–2199PubMedCrossRefGoogle Scholar
  26. Yasuda H, Tada Y, Hayashi Y, Jomori T, Takaiwa F (2005) Expression of the small peptide GLP-1 in transgenic plants. Transgenic Res 14:677–684PubMedCrossRefGoogle Scholar
  27. Zhang J, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V, Gilbertson L (2003) Cre/lox mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor Appl Genet 107:1157–1168PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  1. 1.Functional Transgenic Crops Research Unit, Genetically Modified Organism Research CenterNational Institute of Agrobiological SciencesTsukubaJapan

Personalised recommendations