Generation of CRISPR/Cas9-mediated mutants in Monochasma savatieri using a hairy root system

Monochasma savatieri Franch. ex Maxim is a perennial medicinal plant with antibiotic properties. However, the current M. savatieri resources cannot meet the increasing market demand. Thus, an advanced bioengineering technology platform is required for breeding novel and high-yielding M. savatieri germplasm/cultivars with high value. In the present study, a hairy root system induced by Agrobacterium rhizogenes, which is often used as a model to study root function and bioactive compound production, was established for M. savatieri for the first time. The hairy root induction frequencies of the different explants ranged from 4.6% to 68.3% after infection with A. rhizogenes strain LBA9402. Furthermore, many co-transformed hairy root lines using the binary vector pEG100-35S::GFP were generated. Fluorescence microscopy revealed that GFP was expressed in 72.9% of the transgenic hairy root lines. After six months, the co-transformed hairy root lines were able to grow into trans-genic plantlets on medium containing cytokinin. In addition, to construct a gene editing system for M. savatieri, the tRNA-target-gRNA cassette was cloned into the pRGEB31 vector with AtU6-sgRNA and hSpCas9 backbones. The cellulose synthase-like D (CSLD2/3) genes were used as markers to assess the gene editing efficiency of the CRISPR/Cas9 vector in M. savatieri. The results showed that the CSLD2/3 sequences were altered in 11 out of 59 co-transformed hairy root lines. This study successfully established a genome editing platform for M. savatieri, for the first time, using the A. rhizogenes-mediated co-transformation system. This will enable the development of novel M. savatieri germplasm/cultivars for facility cultivation and exploitation of its medicinal value in the future.