Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens View Full Text


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Article Info

DATE

2006-06

AUTHORS

Yukoh Hiei, Toshihiko Komari

ABSTRACT

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T0) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T0 and T1 generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved. More... »

PAGES

271

References to SciGraph publications

  • 1987-05. Isozymes and classification of Asian rice varieties in THEORETICAL AND APPLIED GENETICS
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  • 1995-05. An improved procedure for plant regeneration from indica and japonica rice protoplasts in PLANT CELL REPORTS
  • 2004-10. Suppression of transfer of non-T-DNA ‘vector backbone’ sequences by multiple left border repeats in vectors for transformation of higher plants mediated by Agrobacterium tumefaciens in MOLECULAR BREEDING
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  • 1996-08. Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties in PLANTA
  • 1991-10. Production of Transgenic Rice (Oryza Sativa L.) Plants from Agronomically Important Indica and Japonica Varieties via Electric Discharge Particle Acceleration of Exogenous DNA into Immature Zygotic Embryos in NATURE BIOTECHNOLOGY
  • 2001-10. Plant regeneration from transformed embryogenic callus of an elite indica rice via Agrobacterium in PLANT CELL, TISSUE AND ORGAN CULTURE (PCTOC)
  • 2003-03. Identification of five new blast resistance genes in the highly blast-resistant rice variety IR64 using a QTL mapping strategy in THEORETICAL AND APPLIED GENETICS
  • 2002-08. Elite Indica Transgenic Rice Plants Expressing Modified Cry1Ac Endotoxin of Bacillus Thuringiensis Show Enhanced Resistance to Yellow Stem Borer (Scirpophaga Incertulas) in TRANSGENIC RESEARCH
  • 1996-09. Agrobacterium-mediated transformation of Javanica rice in MOLECULAR BREEDING
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    http://scigraph.springernature.com/pub.10.1007/s11240-005-9069-8

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    http://dx.doi.org/10.1007/s11240-005-9069-8

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    39 schema:description A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T0) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T0 and T1 generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved.
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