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

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Abstract

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.

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Abbreviations

CSP:

Callus-specific promoter

2,4-D:

2,4-Dichlorophenoxyacetic acid

GFP:

Green fluorescent protein

GM crop:

Genetically modified crop

GUS:

β-Glucuronidase

HPT:

Hygromycin phosphotransferase

Mals:

Mutated acetolactate synthase

MCS:

Multi-cloning site

NAA:

Naphthaleneacetic acid

NiR:

Ferredoxin nitrite reductase

NPT:

Neomycin phosphotransferase

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Acknowledgments

We thank Dr. Taiichi Ogawa (National Institute of Agrobiological Sciences) for providing the vector plasmid harboring the ‘Nipponbare’ NiR gene. This research was supported by the research grant “Genomics and Agricultural Innovation, GMC0003” from the Ministry of Agriculture, Forestry and Fisheries of Japan to F. Takaiwa.

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Correspondence to Fumio Takaiwa.

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Communicated by H. Ebinuma.

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Wakasa, Y., Ozawa, K. & Takaiwa, F. Agrobacterium-mediated co-transformation of rice using two selectable marker genes derived from rice genome components. Plant Cell Rep 31, 2075–2084 (2012). https://doi.org/10.1007/s00299-012-1318-9

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  • DOI: https://doi.org/10.1007/s00299-012-1318-9

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