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A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

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A Simplified Wheat Protoplast Transformation System and Guideline for Avoiding Protein Localization Artifacts.

Leyan Li¹, Shuai Zhong¹, Shuai Liu¹

¹State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, College of Life Science, Henan Agricultural University, Zhengzhou 450046, China.

Plants (Basel, Switzerland)

|June 12, 2026

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Developing a simplified wheat protoplast system improves protein localization studies. Tag position and promoter choice are critical for accurate subcellular localization and phase separation analysis, minimizing experimental artifacts.

Area of Science:

Plant molecular biology
Cell biology
Biochemistry

Background:

Transient protoplast transformation is crucial for wheat protein studies.
Existing methods have limitations, leading to artifacts in localization and phase separation analysis.
Systematic vector design analysis is lacking.

Purpose of the Study:

To establish a simplified and robust wheat mesophyll protoplast transformation method.
To systematically analyze the impact of vector design elements on protein localization and phase separation.
To provide guidelines for minimizing artifacts in wheat research.

Main Methods:

Developed a simplified wheat mesophyll protoplast transformation protocol with a shortened cycle and streamlined handling.
Examined the effects of fluorescent tag position (N- vs. C-terminal) and promoter type (native, single CaMV35S, double CaMV35S).

Keywords:

DNA transformation Triticum aestivum L.fluorescent protein phase separation protoplast subcellular localization transient expression

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Utilized confocal imaging to acquire high-quality data on protein localization and phase separation.

Main Results:

The simplified method enables stable, high-quality confocal imaging data acquisition.
Fluorescent tag position significantly impacts the accuracy of protein localization patterns.
Native promoters are optimal for physiological accuracy; strong constitutive promoters (CaMV35S) can cause overexpression artifacts, leading to false positives or negatives.

Conclusions:

The standardized transformation system minimizes artifacts in wheat protein localization and phase separation research.
Defined vector design principles are essential for reliable experimental outcomes.
This framework enhances the study of protein behavior in wheat.