Targeted Mutagenesis in Tetraploid Switchgrass

Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that is considered as a model biomass crop. However, the self-infertility and high ploidy level of this species make it difficult to study gene function or improve germplasm. Iowa State University's Yang Liu explored the feasibility of using CRISPR-Cas9 for targeted mutagenesis in tetraploid switchgrass.

The team first tested CRISPR in switchgrass with a non-functional green-fluorescent protein gene with an insertion in its 5′ coding region. The gene was successfully mutated by a Cas9/sgRNA complex, resulting in its restored function. Agrobacterium-mediated transformation of calli derived from mature caryopses was then performed, targeting several genes, namely teosinte branched 1 (tb1) a and b and phosphoglycerate mutase (PGM).

Primary transformants (T0) containing CRISPR-Cas9-induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), with the T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was also obtained for the PGM gene. No apparent phenotypical alterations were observed in the PGM T0 mutants.

This study proves that CRISPR-Cas9 system can generate targeted mutagenesis effectively in switchgrass.

For more on this study, read the article in Plant Biotechnology Journal.