Bialaphos selection of stable transformants from maize cell culture

Area of Science:

• Plant Biotechnology
• Genetics
• Molecular Biology

Background:

• Maize (Zea mays) is a vital crop, and efficient genetic transformation methods are crucial for its improvement.
• Developing stable transformed maize callus is a key step for generating genetically modified plants.
• Herbicide resistance is a desirable trait for crop management.

Purpose of the Study:

• To establish a stable transformation protocol for Black Mexican Sweet (BMS) maize using herbicide resistance as a selectable marker.
• To evaluate the expression of both selectable and screenable marker genes in transformed maize callus.
• To assess the correlation between herbicide resistance levels and gene expression.

Main Methods:

• Maize suspension culture cells were bombarded with plasmid DNA containing the bar gene (bialaphos resistance) and a GUS reporter gene.
• Transformed cells were selected on a medium containing bialaphos, which is converted to phosphinothricin (PPT).
• Phosphinothricin acetyl transferase (PAT) activity was assayed by PPT inactivation, and GUS expression was evaluated.

Main Results:

• Stable, bialaphos-resistant BMS maize callus was successfully recovered.
• The presence and expression of the bar gene, encoding PAT, were confirmed in resistant callus.
• Higher PAT expression correlated with faster growth on increasing bialaphos concentrations.
• Fifty percent of the bialaphos-resistant transformants also expressed the non-selected GUS gene.

Conclusions:

• Bialaphos resistance is an effective selection method for maize transformation.
• Co-transformation and expression of non-selected genes (like GUS) occur at a significant frequency.
• This method provides a foundation for developing genetically engineered maize with desirable traits.