African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12400

Full Length Research Paper

A reproducible protocol for regeneration and transformation in canola (Brassica napus L.)

Reda E. A. Moghaieb
  • Reda E. A. Moghaieb
  • Department of Genetics and Genetic Engineering Research Center (GERC), Faculty of Agriculture, Cairo University, Giza, Egypt.
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Mohamed A. El-Awady
  • Mohamed A. El-Awady
  • Department of Genetics and Genetic Engineering Research Center (GERC), Faculty of Agriculture, Cairo University, Giza, Egypt.
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Rabab G. El Mergawy
  • Rabab G. El Mergawy
  • Department of Genetics and Genetic Engineering Research Center (GERC), Faculty of Agriculture, Cairo University, Giza, Egypt.
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SawsanS. Youssef
  • SawsanS. Youssef
  • Department of Genetics and Genetic Engineering Research Center (GERC), Faculty of Agriculture, Cairo University, Giza, Egypt.
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Ahmed M. El-Sharkawy
  • Ahmed M. El-Sharkawy
  • Department of Genetics and Genetic Engineering Research Center (GERC), Faculty of Agriculture, Cairo University, Giza, Egypt.
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  •  Accepted: 05 October 2005
  •  Published: 16 January 2006

Abstract

The objective of the present study is to develop an efficient protocol for shoot and plant regeneration using five commercial canola cultivars grown under the Egyptian agricultural conditions. The regeneration efficiency from hypocotyl explants was examined. The data indicated that embryonic calli were formed within two weeks in the presence of 1 mgl-12,4-D. Adventitious shoots emerged from the embryonic callus in the presence of 4.5 mgl-1 BA. The cultivars showed a varied response to shoot regeneration. Regeneration frequency was high in the cultivar Sarow-4 (68%) followed by Masrri L-16 (64%) compared with the other cultivars tested. Hypocotyl explants from the cultivars Sarow-4 and Semu-249 were inoculated and co-cultivated with Agrobacterium tumefaciensstrain LBA4404 harboring a binary vector pBI-121 containing the neomycin phosphotransferase-II gene (NPT-II). The resulted putative transgenic plantlets were able to grow under knanamycin containing medium. The stable integration of the NPT-IIgene into the plant genomes was tested by PCR using NPT-II -specific primers. The GUS gene expression can be detected only in the transgenic plants. The reported protocol in the present study is repeatable and can be used to regenerate transgenic canola plants expressing the genes present in A. tumifaciens binary vectors.

 

Key words: Agrobacterium, canola, GUS assay, regeneration, fransformation, NPT IIgene.