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Related Experiment Videos

Factors affecting transient expression of vector constructs in wheat protoplasts

K Z Ahmed1, S Omirulleh, F Sági

  • 1Cereal Research Institute, Szeged, Hungary.

Acta Biologica Hungarica
|January 1, 1997
PubMed
Summary

Optimizing polyethylene glycol (PEG) treatment for wheat protoplast transformation enhances transient gene expression. Magnesium ions (Mg2+) and specific culture media significantly boost beta-glucuronidase (GUS) gene activity.

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Area of Science:

  • Plant Biotechnology
  • Molecular Biology
  • Agricultural Science

Background:

  • Efficient gene delivery into plant cells is crucial for crop improvement.
  • Protoplasts offer a viable system for direct DNA uptake but require optimized transformation protocols.
  • Reporter gene systems, like beta-glucuronidase (GUS), are essential for quantifying gene expression efficiency.

Purpose of the Study:

  • To optimize polyethylene glycol (PEG)-mediated direct gene transfer into winter wheat (Triticum aestivum L.) protoplasts.
  • To evaluate the impact of different cations, PEG concentrations, incubation times, and culture media on transient gene expression.
  • To identify highly active promoters for efficient gene expression in wheat protoplasts.

Main Methods:

  • Direct uptake of GUS reporter gene constructs into embryogenic cell suspension culture-derived wheat protoplasts using polyethylene glycol (PEG) treatment.

Related Experiment Videos

  • Optimization of PEG concentration (20%), Mg2+ vs. Ca2+ ions, incubation time (5-10 min at 25°C), and protoplast culture media (KM vs. GM).
  • Quantification of transient GUS expression using spectrofluorimetry and comparison of promoter activities among five different plasmid constructs.

    • Main Results:

    • A 20% PEG concentration with Mg2+ significantly enhanced transient gene expression compared to Ca2+-containing media.
    • Optimal incubation in the transformation mixture was 5-10 minutes at 25°C, with maximum GUS activity observed 48 hours post-uptake.
    • The KM medium supported higher GUS activity than the GM medium, and the pKM794 construct (CaMV 35S promoter with enhancers) showed 6-16 fold higher activity.

    Conclusions:

    • Polyethylene glycol (PEG)-mediated transformation with Mg2+ is an effective method for enhancing transient gene expression in wheat protoplasts.
    • Optimized incubation times and specific culture media (KM) are critical for maximizing reporter gene activity.
    • The pKM794 construct, driven by the CaMV 35S promoter with enhancers, demonstrates superior promoter strength for wheat protoplast transformation.