Functional analysis of theteosinte branched 1gene in the tetraploid switchgrass (Panicum virgatum L.) by CRISPR/Cas9-directed mutagenesis

Abstract

AbstractTillering 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 non-chimericPvtb1aandPvtb1bmutants from chimeric T0 mutants using nodal culture. The biallelicPvtb1a-Pvtb1bmutant plants produced significantly more tillers and higher fresh weight biomass than the wild-type plants. The increased tiller production in the mutant plants resulted primarily from hastened outgrowth of lower axillary buds. Increased tillers were also observed in transgene-free T1 monoallelic mutants for eitherPvtb1a-Pvtb1borPvtb1bgene alone, suggestingPvtb1genes act in a dosage-dependent manner. Transcriptome analysis showed 831 genes were differentially expressed in thePvtb1a-Pvtb1bdouble knockdown mutant. Gene Ontology analysis revealed downregulation ofPvtb1genes affected multiple biological processes, including transcription, flower development, cell differentiation, and stress/defense responses in edited plants. This study demonstrates thatPvtb1genes 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.HighlightSolid non-chimeric mutants were successfully isolated from CRISPR/Cas9-induced chimeric mutants using nodal culture.Teosinte branched 1(tb1) genes are involved in various pathways to regulate tillering in switchgrass.