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A Robotic Platform for High-throughput Protoplast Isolation and Transformation.

Elizabeth M Dlugosz1, Scott C Lenaghan2, C Neal Stewart1

  • 1Department of Plant Sciences, University of Tennessee, Knoxville.

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|October 22, 2016
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This study presents an automated, robotic protocol for plant protoplast isolation and transformation, significantly reducing processing time to under 4 hours. This advancement aims to accelerate plant genomics research by enabling high-throughput analysis of plant gene expression and editing.

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Area of Science:

  • Plant Science
  • Molecular Biology
  • Genomics

Background:

  • Plant protoplast research is experiencing a resurgence for applications in signal transduction, gene regulation, and genome editing.
  • Advances in plant regeneration from protoplasts have increased interest in their use for plant genomics.
  • High-throughput automated protoplast analysis is hindered by the cost of lab-grade enzymes.

Purpose of the Study:

  • To develop an automated protocol for plant protoplast isolation and transformation using a robotic platform.
  • To validate the transformation procedure using a reporter gene and assess protoplast production efficiency.
  • To utilize low-cost, food-grade enzymes for protoplast isolation to reduce costs for high-throughput applications.

Main Methods:

  • Developed an automated protocol for protoplast isolation and transformation from tobacco (BY-2) cell cultures using a robotic platform.
  • Validated transformation efficiency using an orange fluorescent protein (OFP) reporter gene under the Cauliflower mosaic virus 35S promoter.
  • Assessed protoplast production efficiency using propidium iodide staining and employed food-grade enzymes for isolation.

Main Results:

  • Successfully automated protoplast isolation and transformation from BY-2 tobacco cell cultures.
  • Confirmed OFP expression in transformed protoplasts via epifluorescence microscopy.
  • Achieved a complete protoplast isolation to transformation workflow in under 4 hours with no operator input, using cost-effective enzymes.

Conclusions:

  • The developed automated protocol significantly accelerates plant protoplast isolation and transformation.
  • The use of low-cost, food-grade enzymes makes high-throughput automated plant genomics more accessible.
  • The protocol is expected to be translatable to various plant suspension cultures, advancing crop genomics research.