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Stable transformation of rice (Oryza sativa L.) via microprojectile bombardment of highly regenerative, green tissues derived from mature seed

  • Genetic Transformation and Hybridization
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Abstract

A highly efficient and reproducible transformation system for rice (Oryza sativa L. cv. Taipei 309) was developed using microprojectile bombardment of highly regenerative, green tissues. These tissues were induced from mature seeds on NB-based medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and high concentrations of cupric sulfate under dim light conditions; germinating shoots and roots were completely removed. Highly regenerative, green tissues were proliferated on the same medium and used as transformation targets. From 431 explants bombarded with transgenes [i.e. a hygromycin phosphotransferase (hpt) gene plus one of a wheat thioredoxin h (wtrxh), a barley NADP-thioredoxin reductase (bntr), a maize Mutator transposable element (mudrB) or β-glucuronidase (uidA; gus) gene], 28 independent transgenic events were obtained after an 8- to 12-week selection period, giving a 6.5% transformation frequency. Of the 28 independent events, 17 (61%) were regenerable. Co-transformation of the second introduced transgene was detected in 81% of the transgenic lines tested. Stable integration and expression of the foreign genes in T0 plants and T1 progeny were confirmed by DNA hybridization, western blot analyses and germination tests.

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Abbreviations

2,4-D :

2,4-Dichlorophenoxyacetic acid

BAP :

6-Benzylaminopurine

BNTR :

Barley NADP-thioredoxin reductase

DTNB :

2′,5′-Dithiobis-(2-nitrobenzoic acid)

HPT :

Hygromycin phosphotransferase

IE :

Immature embryos

MS :

Murashige and Skoog

PCR :

Polymerase chain reaction

uidA :

β-Glucuronidase gene

WTRX h :

Wheat thioredoxin h

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Acknowledgements

M.-J. Cho, D. Okamoto, H.-K. Kim, H.-R. Jung, K. Newcomb and H.S. Yoo were supported by funds from the BioSTAR Program of the University of California and Ventria Bioscience of Sacramento, Calif. P.G. Lemaux was supported by the USDA Cooperative Extension Service through the University of California. B.B. Buchanan acknowledges support from the California Agricultural Experiment Station.

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Authors and Affiliations

  1. Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA

    M.-J. Cho, H. Yano, D. Okamoto, H.-K. Kim, H.-R. Jung, K. Newcomb, V. K. Le, H. S. Yoo, R. Langham, B. B. Buchanan & P. G. Lemaux

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  1. M.-J. Cho
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  2. H. Yano
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  3. D. Okamoto
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  4. H.-K. Kim
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  5. H.-R. Jung
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  6. K. Newcomb
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  7. V. K. Le
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  8. H. S. Yoo
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  9. R. Langham
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  10. B. B. Buchanan
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  11. P. G. Lemaux
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Corresponding author

Correspondence to M.-J. Cho.

Additional information

Communicated by I.S. Chung

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Cho, MJ., Yano, H., Okamoto, D. et al. Stable transformation of rice (Oryza sativa L.) via microprojectile bombardment of highly regenerative, green tissues derived from mature seed. Plant Cell Rep 22, 483–489 (2004). https://doi.org/10.1007/s00299-003-0713-7

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  • DOI: https://doi.org/10.1007/s00299-003-0713-7

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