Protocol for efficient ginseng transformation

Abstract

Panax ginseng is a major medicinal crop with pharmaceutical e cacy derived from ginsenoside metabolites. Despite its genome information, the ine ciency of ginseng transformation hinders the study of the molecular mechanism of ginseng plant metabolism. Thus, this protocol aimed to develop an easy and e cient system for ginseng transformation. We established a transformation system using ginseng callus cultured in a liquid medium, which has a higher ratio compared with cotyledon explant. In addition, Agrobacterium tumefaciens has been used for plant transformation. Compared with the LBA4404 strain, C58C1 was inappropriate for ginseng transformation using ginseng callus. We induced and maintained calli in liquid medium and cut them into small pieces before infection. After infection, we selected calli that survived from the antibiotic medium until identi cation of newly growing cells. In β-glucuronidase (GUS) gene assay, the expression of the GUS gene was observed in cells that were newly generated from explants. We treated calli with 0.05 M of MgSO 4 before infection. After MgSO 4 pre-treatment, the transformation e ciency of growing cells around infected callus was increased. Moreover, we constructed and introduced a visible reporter RUBY system to easily identify transformed cells. Using this system, we could identify the cells by a red color with naked eyes. Based on our transformation protocol, the success rate has increased to 77.27% in surviving lines during selection culture. This stable ginseng transformation could facilitate the overexpression and knockout of ginseng lines for functional or synthetic biological studies. Key MessageWe re-designed the ginseng transformation protocol using ginseng calli for explant material, and MgSO 4 pretreatment was conducted before Agrobacterium infection. RUBY reporter was successfully introduced in ginseng.