| Authors: | Cheng, Q., Dong, L., Su, T., Li, T., Gan, Z., Nan, H., Lu, S., Fang, C., Kong, L., Li, H., Hou, Z., Kou, K., Tang, Y., Lin, X., Zhao, X., Chen, L., Liu, B., Kong, F. |
| Abstract: | 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 fourLATE 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 usingAgrobacterium rhizogenesstrain K599. Once confirmed, we performed stable soybean transformation and obtained 19 independent transgenic soybean plants. Subsequently, we obtained one T1transgene-free homozygous quadruple mutant ofGmLHYby self-crossing. The phenotypes of the T2-generation transgene-free quadruple mutant plants were observed, and the results showed that the quadruple mutant ofGmLHYdisplayed reduced plant height and shortened internodes. The levels of endogenous gibberellic acid (GA3) inGmlhy1a1b2a2bwas 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 ofGmLHYwere significantly decreased in comparison to the WT. These results suggest thatGmLHYencodes 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 fourGmLHYgenes reduces soybean plant height and shortens internodes from 20 to 35days after emergence (DAE). These findings provide insight into the mechanisms underlying plant height regulatory networks in soybean. |
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