Abstract: In order to explore the possible application of CRISPR/Cas9 gene editing technology in peanut, the genetic transformation by pollen tube injection pathway and Agrobacterium tumefaciens mediated method was conducted firstly in peanut. The transformation efficiency was improved dramatically by adding appropriate concentration of AS, MES and MgCl₂ · 6H₂O to the infection solution, and using the fresh infection solution produced daily which could maximize the activity of A. tumefaciens.In this experiment, the transformation method was applied to peanut gene editing based on CRISPR/Cas9 technology for the first time. The editing target site was designed according to the peanut FatB gene sequence, and the CRISPR/Cas9 gene editing vector PX458-Cas9-FatB was successfully constructed and transformed into the A. tumefaciens strain GV3101. The fresh A. tumefaciens infection solution was injected into the carina of peanut plants by 1 mL aseptic syringe until the petals were soaked. The injection was conducted before 8:00 everyday, with 15 d continuous injection. Those pod needles from injected flowers were then marked by tied with nylon cord. Those pods tied by nylon cord from injected flowers were harvested, and then dried by normal sunlight. The genomic DNAs from those seeds from marked pods were extracted,and the positive transformed seeds were screened by PCR amplification.The results showed that among the 274 seeds tested, the corresponding target bands were obtained from 108 seeds, and the positive rate of transformation was 39.42%.The sequencing results showed that one of the 108 positive kernels had gene editing outside the target site, the kernels with edited target sites were found in the inbred progenies of some positive kernels. Therefore, this study preliminarily showed that the injection dipping flower method was effective for peanut gene editing based on CRISPR/Cas9 technology, but the editing efficiency needs to be further improved.

Key words: Peanut, CRISPR/Cas9, Infection and soaking flower, FatB, Gene editing

CLC Number: 

• Q78

• S565.03