Abstract
Weeds cause the largest yield loss in soybean production. The development of herbicide-resistant soybean germplasm is of great significance for weed control and yield improvement. In this study, we used the cytosine base editor (BE3) to develop novel herbicide-resistant soybean. We have successfully introduced base substitutions in GmAHAS3 and GmAHAS4 and obtained a heritable transgene-free soybean with homozygous P180S mutation in GmAHAS4. The GmAHAS4 P180S mutants have apparent resistance to chlorsulfuron, flucarbazone-sodium, and flumetsulam. In particular, the resistance to chlorsulfuron was more than 100 times that of with wild type TL-1. The agronomic performance of the GmAHAS4 P180S mutants showed no significant differences to TL-1 under natural growth conditions. In addition, we developed allele-specific PCR markers for the GmAHAS4 P180S mutants, which can easily discriminate homozygous, heterozygous mutants, and wild-type plants. This study demonstrates a feasible and effective way to generate herbicide-resistant soybean by using CRISPR/Cas9-mediated base editing.