Abstract
BackgroundSoybean (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.ResultsHere, 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 T1 transgene-free homozygous quadruple mutant of GmLHY by self-crossing. The phenotypes of the T2-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.ConclusionsOur 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.
Highlights
IntroductionPlant height is a key trait that significantly impacts the yield of soybean; research on the molecular mechanisms associated with soybean plant height is lacking
Soybean (Glycine max) is an economically important oil and protein crop
Phylogenetic analysis showed that the four CCA1/LATE ELONGATED HYPOCOTYL (LHY) orthologs are closer to AtLHY than AtCCA1
Summary
Plant height is a key trait that significantly impacts the yield of soybean; research on the molecular mechanisms associated with soybean plant height is lacking. Soybean is one of the most important economic sources of vegetable oil and protein worldwide, and plant height, node number, internode length, branch number, and seed size are significant factors that affect soybean yield [1, 2]. Some plant height genes have been cloned by map-based cloning in several plant species, such as maize [6,7,8], rice [9,10,11], tomato [12], and soybean [13, 14]. The Brachytic (Br2) gene, which was cloned from maize by mapping, significantly impacts plant height [8]. OsNAC2 is a NAC transcription factor, and the constitutive expression of OsNAC2 resulted in shorter internodes and shorter spikelets in rice [15]
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