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High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola (Brassica napus)

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Abstract

Five different varieties of Brassica napus (Cyclone, Dunkled, Oscar, Rainbow and KS75) were tested for their regeneration response. Cyclone showed a very high frequency of regeneration (92%). The use of silver nitrate was a pre-requisite for efficient shoot regeneration. Hypocotyls were selected as the starting material for transformation experiments on the basis of high transient GUS expression. Explants were co-cultivated with Agrobacterium strain EHA101 harboring a binary vector pIG121Hm containing neomycin phosphotransferase II (NPTII) gene, conferring resistance to kanamycin, hygromycin phosphotransferase (HPT) gene, conferring resistance to hygromycin as selectable markers and β-glucuronidase (GUS) gene as a reporter. Acetosyringone promoted the transformation but was not an absolute requirement. A pre-selection period of 7 days after co-cultivation was essential for successful transformation. Kanamycin was efficient selective agent for selection and maximum transformation efficiency was 24%. GUS activity was evident in leaf tissues. All the transgenic plants have an expected band of 0.43 kb fragment by PCR analysis confirming the presence of foreign DNA into plant genome.

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Authors and Affiliations

  1. National Agricultural Research Centre, Agricultural Biotechnology Institute, Park Road, Islamabad, Pakistan

    Muhammad Ramzan Khan, Hamid Rashid & Zubeda Chaudry

  2. Department of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan

    Muhammad Ansar

Authors
  1. Muhammad Ramzan Khan
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  2. Hamid Rashid
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  3. Muhammad Ansar
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  4. Zubeda Chaudry
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Correspondence to Muhammad Ramzan Khan.

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Ramzan Khan, M., Rashid, H., Ansar, M. et al. High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola (Brassica napus). Plant Cell, Tissue and Organ Culture 75, 223–231 (2003). https://doi.org/10.1023/A:1025869809904

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