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Scalable Transfection of Maize Mesophyll Protoplasts
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Protoplast Isolation and Transfection in Maize.

Monique R Coy1, Shane E Abbitt1, Mary J Frank2

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Methods in Molecular Biology (Clifton, N.J.)
|March 8, 2022
PubMed
Summary

This study presents efficient protocols for isolating and transfecting maize mesophyll protoplasts for gene expression studies. These methods yield high numbers of viable protoplasts and achieve significant transfection efficiency within 24 hours.

Keywords:
Dual-expression vectorFluorescent proteinLeafMesophyllPolyethylene glycolTransientZea maize

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Area of Science:

  • Plant molecular biology
  • Gene expression analysis
  • Maize genetics

Background:

  • Protoplast-based transient gene expression is vital for studying gene regulation.
  • Efficient isolation of healthy protoplasts is critical for successful gene expression studies.
  • Existing methods may require complex steps for normalization and analysis.

Purpose of the Study:

  • To describe optimized protocols for isolating and transfecting maize mesophyll protoplasts.
  • To enable robust gene expression studies in maize.
  • To provide a streamlined workflow for protoplast-based assays.

Main Methods:

  • Isolation of maize mesophyll protoplasts from etiolated leaf tissue.
  • Polyethylene glycol (PEG)-mediated transfection of protoplasts.
  • Utilization of a dual-fluorescent protein (FP) expression vector for normalization and gene of interest expression.

Main Results:

  • Isolation protocol yields 1.8-1.9 × 10^7 protoplasts with 80-90% viability from 6g of tissue.
  • Transfection protocol achieves 55-58% transfection efficiency.
  • Dual-FP vector simplifies normalization and eliminates need for separate enzymatic assays.

Conclusions:

  • The described protocols provide an efficient and reliable method for maize protoplast isolation and transfection.
  • The dual-FP vector system streamlines gene expression analysis and improves accuracy.
  • These protocols are applicable to maize genotypes B73 and PHR03 and can be completed within 24 hours.