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Updated: Mar 21, 2026

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Published on: September 27, 2016

Efficient production of Aschersonia placenta protoplasts for transformation using optimization algorithms.

Xiuyan Wei^1,2,3, Xinyue Song^1,2,3, Dong Dong^1,2,3

¹a Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.

Canadian Journal of Microbiology

|May 19, 2016

Summary

This study optimized conditions for producing transformable Aschersonia placenta protoplasts, yielding 10-fold more than non-optimized methods. These advancements enable genetic studies of this important insect pathogenic fungus.

Keywords:

Aschersonia placenta condition de production méthodologie des surfaces de réponses production condition protoplast yield rendement de protoplastes response surface method

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Last Updated: Mar 21, 2026

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Published on: September 27, 2016

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

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Published on: January 25, 2018

Scalable Transfection of Maize Mesophyll Protoplasts

Scalable Transfection of Maize Mesophyll Protoplasts

Published on: June 23, 2023

Area of Science:

Mycology
Biotechnology
Insect Pathology

Background:

Aschersonia placenta is an effective pathogen of whiteflies and scale insects.
Limited genetic tools hinder the study of A. placenta.
Developing efficient genetic manipulation methods is crucial for understanding this fungus.

Purpose of the Study:

To optimize the production of transformable Aschersonia placenta protoplasts.
To establish a reliable protocol for generating high yields of viable protoplasts.
To facilitate downstream genetic applications for A. placenta.

Main Methods:

Utilized Response Surface Methodology (RSM) with a Box-Behnken Design (BBD) to model critical parameters for protoplast production.
Optimized variables included lywallzyme concentration, digestion time, and recovery time in a NaCl-Tris buffer.
Verified the model's predictive ability with R(2) = 0.9465.

Main Results:

Achieved a maximal protoplast yield of (4.41 ± 0.02) × 10(7) cells/mL under optimized conditions.
Optimized conditions involved 26.1 mg/mL lywallzyme, 4 h digestion, and 64.6 h recovery in 0.7 mol/L NaCl-Tris buffer.
Protoplast yield was approximately 10-fold higher compared to non-optimized methods.
Demonstrated successful transformation with vector PbarGPE, achieving an efficiency of 300 colonies/(μg DNA·10(7) protoplasts).
Confirmed vector DNA integration via PCR.

Conclusions:

Rational design strategies, specifically RSM and BBD, are effective for enhancing fungal protoplast production.
The developed protocol significantly increases protoplast yield, enabling genetic studies of A. placenta.
These optimized protoplasts are suitable for genetic transformation, paving the way for further research and application of this insect pathogenic fungus.