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

DOI: 10.1007/s00299-012-1318-9

Cite this article as:
Wakasa, Y., Ozawa, K. & Takaiwa, F. Plant Cell Rep (2012) 31: 2075. doi:10.1007/s00299-012-1318-9


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 synthaseAgrobacteriumCo-transformationFerredoxin nitrite reductaseGM cropTransgenic 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

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

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