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In planta transformation in wheat: an improved protocol to develop wheat transformants.

Satish Kumar1, Harinder Vishwakarma1, Gourab Ghosh1

  • 1ICAR-National Institute for Plant Biotechnology, Pusa Campus, New Delhi, 110012, India.

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|March 9, 2024
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Summary

Researchers developed a simpler, faster wheat transformation method. Early screening of transgenic plants (T0) reduced workload without losing efficiency, achieving 3.33% transformation in wheat genotype HD2894.

Keywords:
GFP analysisIn-planta transformationPCRShoot apical meristemSouthern hybridizationWheat

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Area of Science:

  • Plant Biotechnology
  • Molecular Biology
  • Agricultural Science

Background:

  • Efficient transformation protocols are crucial for wheat genome editing and transgenic development.
  • Previous in planta transformation methods for Indian bread wheat were labor-intensive and time-consuming.
  • This study aimed to improve the in planta transformation method for simplicity, efficiency, and reduced labor.

Purpose of the Study:

  • To develop a simplified and efficient in planta transformation protocol for wheat.
  • To reduce the labor and time required for wheat transformation.
  • To establish a method for generating transgenic and genome-edited wheat lines.

Main Methods:

  • Inoculation of wheat seedling shoot apical meristem with Agrobacterium tumefaciens harboring the GFP gene.
  • Introduction of PCR-based screening for transgene-positive plants at the T0 stage.
  • Analysis of T1 generation plants for stable transgene integration using PCR and fluorescent microscopy.

Main Results:

  • PCR screening at the T0 stage identified 27 transgene-positive plants.
  • Out of 6 PCR-positive T1 plants, 4 showed stable transgene integration (GFP).
  • Fluorescent microscopy confirmed GFP expression in the 4 Southern hybridization-positive T1 plants.

Conclusions:

  • T0 stage screening effectively reduced the number of plants for subsequent generations without compromising transformation efficiency.
  • A simple, efficient, and less labor-intensive method achieved 3.33% transformation efficiency in wheat genotype HD2894.
  • The improved method facilitates the development of transgenic and genome-edited wheat with desirable traits.