Targeted mutagenesis in wheat microspores using CRISPR/Cas9

Area of Science:

• Plant Science
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 gene editing accelerates crop development but is limited by long crop life cycles.
• Microspore technology generates homozygous plants rapidly, offering a solution for efficient breeding.

Purpose of the Study:

• To develop an optimized haploid mutagenesis system combining CRISPR/Cas9 and microspore technology for wheat genetic modification.
• To identify optimal conditions for delivering CRISPR/Cas9 reagents into wheat microspores.

Main Methods:

• Investigated factors affecting CRISPR/Cas9 reagent delivery into microspores.
• Utilized electroporation with the Neon transfection system, optimizing cell number, DNA amount, and voltage.
• Employed multiple Cas9 and sgRNA constructs for targeted gene modifications.

Main Results:

• Determined optimal conditions for microspore transfection: minimum 75,000 cells, 10-20 µg DNA, and 500 V pulsing voltage.
• Successfully introduced targeted modifications into an exogenous DsRed gene and endogenous wheat genes (TaLox2, TaUbiL1).

Conclusions:

• The combined CRISPR/Cas9 and microspore technology system is effective for inducing targeted genetic modifications in wheat.
• This approach significantly enhances trait discovery and crop improvement by accelerating the generation of desired genotypes.