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[Agrobacterium tumefaciens mediated maize transformation].

L Huang1, Z M Wei

  • 1Shanghai Institute of Plant Physiology, The Chinese Academy of Sciences, Shanghai 200032.

Shi Yan Sheng Wu Xue Bao
|January 29, 2003
PubMed
Summary

Researchers successfully transformed maize plants using Agrobacterium tumefaciens, achieving stable gene integration. However, some transgenic calli lost GUS gene activity due to high DNA methylation, impacting regeneration ability.

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Area of Science:

  • Plant Biotechnology
  • Molecular Biology
  • Genetics

Background:

  • Agrobacterium tumefaciens-mediated transformation is a key tool for genetic modification in crops.
  • Understanding transgene integration and expression is crucial for developing improved maize varieties.

Purpose of the Study:

  • To develop transgenic maize plants with stable T-DNA integration.
  • To investigate the expression of the GUS reporter gene and its stability in transgenic maize.
  • To analyze factors affecting transgene stability and regeneration in long-term cultures.

Main Methods:

  • Maize inbreds were transformed using Agrobacterium tumefaciens EHA101 (pGIH).
  • Polymerase Chain Reaction (PCR) and Southern blot analyses were used to confirm T-DNA integration.
  • GUS gene expression was assessed using X-gluc staining.
  • Inverse PCR determined transgene copy number.
  • HpaII digestion and Southern blot analysis investigated DNA methylation patterns.

Main Results:

  • Successful transformation of maize inbreds, with Suyu No. 1 showing an 8.1% transformation frequency.
  • Stable integration of T-DNA into the maize genome was confirmed by PCR and Southern blot.
  • GUS gene expression was detected in maize cells.
  • Transgenic lines exhibited a single copy of the transgene.
  • Some hygromycin-resistant calli lost regeneration ability and GUS activity over time, despite confirmed gusA gene integration.
  • Southern blot analysis indicated high methylation of the transgenic gusA gene in non-expressing calli.

Conclusions:

  • Agrobacterium-mediated transformation is effective for creating transgenic maize with stable transgene integration.
  • Post-integration epigenetic modifications, specifically DNA methylation, can lead to gene silencing and loss of desirable traits in transgenic plants.
  • Further research is needed to understand and control epigenetic modifications for enhanced stability of transgene expression in maize.

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