Abstract
Background: Soybean ( Glycine max ) is an important economically crops for plant oil and protein in the world. The plant height as a key trait has significant effects on yield of soybean , however, the research on molecular mechanism for soybean plant height is still unclear. Recently, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated) system as a new technology for gene editing, has been rapidly utilized to edit the genomes of crop plants. Results: Here, we designed four gRNAs to mutate four LATE ELONGATED HYPOCOTYL ( LHY ) / CIRCADIAN CLOCK ASSOCIATED1 ( CCA1 )- LIKE ( LCL ) 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 rhizogenesis strain K599. Once confirmed, we performed stable soybean transformation and obtained nineteen independent transgenic soybean plants. Subsequently, we obtained one T 1 transgene-free homozygous quadruple mutant of GmLCL by self-crossed. The phenotype of T 2 -generation transgene-free quadruple mutant plants were observed and the results showed that quadruple mutant of GmLCL displayed reduced plant height and shortened internodes. In addition, the relative expression levels of gibberellic acid (GA) metabolic pathway genes in the quadruple mutant of GmLCL were significantly decreased than wild type (WT). It suggests that GmLCLs encoding MYB transcription factor affect plant height through mediating the GA pathway in soybean. We also develop some genetic markers to identify mutant for assisting breeding studies. Conclusions: Our results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four GmLCL genes reduce soybean plant height and shorten internodes. These findings suggest that manipulation of four GmLCL genes may improve yield by altered plant height and internode length in soybean.
Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants