Plant Cell Reports

, Volume 27, Issue 2, pp 319–327

Genetic transformation of NERICA, interspecific hybrid rice between Oryza glaberrima and O. sativa, mediated by Agrobacterium tumefaciens

Genetic Transformation and Hybridization

DOI: 10.1007/s00299-007-0465-x

Cite this article as:
Ishizaki, T. & Kumashiro, T. Plant Cell Rep (2008) 27: 319. doi:10.1007/s00299-007-0465-x

Abstract

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T0 and T1 generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.

Keywords

Agrobacterium tumefaciens  NERICA  Transformation 

Abbreviations

2,4-D

2,4-Dichlorophenoxyacetic acid

35S

Cauliflower mosaic virus 35S

GUS

β-Glucuronidase

HPT

Hygromycin phosphotransferase

NAA

Naphthaleneacetic acid

NERICA

New rice for Africa

NOS

Nopaline synthase

NPTII

Neomycin phosphotransferase II

PCR

Polymerase chain reaction

UBI

Maize ubiquitin

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Biological Resources DivisionJapan International Research Center for Agricultural Sciences (JIRCAS)TsukubaJapan
  2. 2.Tropical Agriculture Research FrontJIRCASIshigakiJapan

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