promoter-driven clustered regularly interspaced short palindromic repeats/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by -mediated transformation.

-mediated (ARM) transformation is an efficient and powerful tool to generate transgenic roots to study root-related biology. For loss-of-function studies, transgenic-root-induced indel mutations by CRISPR/Cas9 only with homozygous/biallelic mutagenesis can exhibit mutant phenotype(s) (excluding recessive traits). However, a low frequency of homozygous mutants was produced by a constitutive promoter to drive expression. Here, we identified a highly efficient gamma- promoter, termed , with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species. achieved higher homozygous/biallelic mutation efficiency than the most widely used promoter in driving expression in soybean, , and tomato roots. Using the p-Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p-Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the p-Cas9 system used in ARM transformation, especially its great potential in diploids with multiple-copy genes targeted mutations and polyploid plants with multiplex genome editing. is conservatively active in various eudicots species, suggesting that might be applied in a wide range of dicots species.