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

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.