Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9

Area of Science:

• Plant Biotechnology
• Molecular Genetics
• Crop Science

Background:

• Switchgrass (Panicum virgatum L.) is a vital biomass crop, but its self-infertility and polyploidy hinder genetic studies and improvement.
• Targeted mutagenesis is crucial for understanding gene function and enhancing crop traits in switchgrass.

Purpose of the Study:

• To assess the efficacy of the CRISPR/Cas9 system for targeted mutagenesis in tetraploid switchgrass.
• To develop methods for efficient gene editing in switchgrass for germplasm improvement.

Main Methods:

• A transient assay using a modified green-fluorescent protein gene was established to validate CRISPR/Cas9 activity.
• Agrobacterium-mediated transformation was employed to introduce CRISPR/Cas9 components targeting teosinte branched 1 (tb1) and phosphoglycerate mutase (PGM) genes.
• Single and dual guide RNA constructs were utilized to induce mutations in selected switchgrass genes.

Main Results:

• CRISPR/Cas9 successfully restored function to the mutated green-fluorescent protein gene in a transient assay.
• Stable transformation yielded mutations in tb1a, tb1b, and PGM genes, with high efficiency for tb1a (95.5%).
• T0 switchgrass mutants for tb1a and tb1b exhibited increased tiller production; PGM mutants showed no apparent phenotypic changes.

Conclusions:

• The CRISPR/Cas9 system is a powerful and effective tool for targeted mutagenesis in tetraploid switchgrass.
• CRISPR/Cas9 facilitates the generation of targeted homozygous mutants in the T0 generation, overcoming limitations of inbreeding.
• This technology holds significant potential for accelerating genetic improvement and functional genomics research in switchgrass.