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Background  Soybean ( Glycine max ) is an economically important oil and protein crop. Plant height is a key trait that significantly impacts the yield of soybean; however, research on the molecular mechanisms associated with soybean plant height is lacking. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated system 9) system is a recently developed technology for gene editing that has been utilized to edit the genomes of crop plants.    Results  Here, we designed four gRNAs to mutate four  LATE ELONGATED HYPOCOTYL    (   LHY ) genes in soybean. In order to test whether the gRNAs could perform properly in transgenic soybean plants, we first tested the CRISPR construct in transgenic soybean hairy roots using  Agrobacterium rhizogenes  strain K599. Once confirmed, we performed stable soybean transformation and obtained 19 independent transgenic soybean plants. Subsequently, we obtained one T 1  transgene-free homozygous quadruple mutant of  GmLHY  by self-crossing. The phenotypes of the T 2 -generation transgene-free quadruple mutant plants were observed, and the results showed that the quadruple mutant of  GmLHY  displayed reduced plant height and shortened internodes. The levels of endogenous gibberellic acid (GA3) in  Gmlhy1a1b2a2b  was lower than in the wild type (WT), and the shortened internode phenotype could be rescued by treatment with exogenous GA3. In addition, the relative expression levels of GA metabolic pathway genes in the quadruple mutant of  GmLHY  were significantly decreased in comparison to the WT. These results suggest that  GmLHY  encodes an MYB transcription factor that affects plant height through mediating the GA pathway in soybean. We also developed genetic markers for identifying mutants for application in breeding studies.    Conclusions  Our results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four  GmLHY  genes reduces soybean plant height and shortens internodes from 20 to 35 days after emergence (DAE). These findings provide insight into the mechanisms underlying plant height regulatory networks in soybean.

CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean