Functional Analysis of the Gene in the Tetraploid Switchgrass (.) by CRISPR/Cas9-Directed Mutagenesis.

Tillering is an important biomass yield component trait in switchgrass (.). ()/ () gene is a known regulator for tillering/branching in several plant species; however, its role on tillering in switchgrass remains unknown. Here, we report physiological and molecular characterization of mutants created by CRISPR/Cas9. We successfully obtained nonchimeric and mutants from chimeric T0 mutants using nodal culture. The biallelic mutant plants produced significantly more tillers and higher fresh weight biomass than the wild-type plants. The increased tiller number in the mutant plants resulted primarily from hastened outgrowth of lower axillary buds. Increased tillers were also observed in transgene-free BC1 monoallelic mutants for either or gene alone, suggesting genes act in a dosage-dependent manner. Transcriptome analysis showed 831 genes were differentially expressed in the a- double knockdown mutant. Gene Ontology analysis revealed downregulation of genes affected multiple biological processes, including transcription, flower development, cell differentiation, and stress/defense responses in edited plants. This study demonstrates that genes play a pivotal role in tiller production as a negative regulator in switchgrass and provides opportunities for further research aiming to elucidate the molecular pathway regulating tillering in switchgrass.