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Tillering is an important biomass yield component trait in switchgrass ( Panicum virgatum L .).  Teosinte branched 1  ( tb1 )/ Branched 1  ( BRC1 ) 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  Pvtb1a  and  Pvtb1b  mutants from chimeric T0 mutants using nodal culture. The biallelic  Pvtb1a-Pvtb1b  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  Pvtb1a-Pvtb1b  or  Pvtb1b  gene alone, suggesting  Pvtb1  genes act in a dosage-dependent manner. Transcriptome analysis showed 831 genes were differentially expressed in the  Pvtb1 a- Pvtb1b  double knockdown mutant. Gene Ontology analysis revealed downregulation of  Pvtb1  genes affected multiple biological processes, including transcription, flower development, cell differentiation, and stress/defense responses in edited plants. This study demonstrates that  Pvtb1  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.

Functional Analysis of the teosinte branched 1 Gene in the Tetraploid Switchgrass (Panicum virgatum L.) by CRISPR/Cas9-Directed Mutagenesis