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A simple and efficient agroinfiltration method for transient gene expression in Citrus.

Yosvanis Acanda1, Stacy Welker1,2, Vladimir Orbović1

  • 1Citrus Research and Education Center, University of Florida/IFAS, Lake Alfred, FL, USA.

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|May 5, 2021
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Summary

Microwounding enhances Agrobacterium infiltration for transient gene expression in citrus. This simple method improves efficiency in difficult-to-transform citrus varieties, aiding functional genomics research.

Keywords:
Citrus agroinfiltrationMicroneedle rollerTransient gene expression

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Area of Science:

  • Plant Biotechnology
  • Molecular Biology
  • Agricultural Science

Background:

  • Agrobacterium infiltration is crucial for transient gene expression studies in plants.
  • Its application in citrus is limited due to low efficiency, hindering functional genomic research.

Purpose of the Study:

  • To develop an efficient, cost-effective, and reliable method for Agrobacterium-mediated transient gene expression in citrus.
  • To optimize parameters for enhanced agroinfiltration in recalcitrant citrus varieties.

Main Methods:

  • Utilized a microneedle roller to create micro-wounds on citrus leaf epidermis.
  • Optimized wound density, leaf maturity, Agrobacterium strain (GV2260), and incubation period (8-10 days).
  • Assessed transient expression levels of Green Fluorescent Protein (GFP).

Main Results:

  • Microwounding significantly improved agroinfiltration efficiency and transient gene expression.
  • Optimal conditions included high wound density, young expanded leaves, strain GV2260, and 8-10 day incubation.
  • Achieved up to 100 ng/mg of GFP expression, enabling successful protein subcellular localization.

Conclusions:

  • Microwounding is a facile and effective pre-treatment for enhancing Agrobacterium infiltration in citrus.
  • This protocol is applicable across multiple citrus varieties and potentially other species with waxy cuticles.
  • Facilitates rapid functional genomic studies in citrus.