Targeted Mutagenesis in Hexaploid Bread Wheat Using the TALEN and CRISPR/Cas Systems

Area of Science:

• Agricultural Science
• Molecular Biology
• Genetics

Background:

• Sequence-specific nucleases like TALENs and CRISPR/Cas9 are vital for reverse genetics.
• These tools facilitate targeted genome modification across various organisms.
• Previous studies have utilized these systems to create loss-of-function alleles in bread wheat.

Purpose of the Study:

• To present detailed protocols for wheat genome modification using TALEN and CRISPR/Cas9 systems.
• To outline methods for designing and constructing target sites for both gene editing systems.
• To describe procedures for evaluating nuclease activity, plant transformation, and mutation screening.

Main Methods:

• Design and construction of TALEN and CRISPR/Cas9 target sites.
• Evaluation of nuclease activity in wheat protoplasts.
• Agrobacterium-mediated plant transformation.
• Molecular screening for induced mutations.

Main Results:

• Established protocols for efficient genome editing in bread wheat.
• Demonstrated the application of both TALEN and CRISPR/Cas9 for generating loss-of-function alleles.
• Provided a comprehensive workflow from target design to mutation identification.

Conclusions:

• TALEN and CRISPR/Cas9 systems offer powerful tools for targeted wheat genome editing.
• The presented protocols facilitate the generation of loss-of-function alleles for genetic studies.
• These methods are crucial for advancing functional genomics and crop improvement in bread wheat.