Establishment of an Agrobacterium-mediated genetic transformation system for Cymbidium faberi Rolfe

Background: Cymbidium faberi Rolfe is an ornamental orchid of high economic value, however, an efficient genetic transformation system has not yet been reported for this species. Aims: This study aimed to establish an Agrobacterium tumefaciens-mediated genetic transformation system for C. faberi and to optimize the key factors affecting transformation performance. Methods: Rhizomes, calli, and inflorescences were used as explants. The pCAMBIA3301-EGFP vector was introduced using Agrobacterium strain GV3101. Explant type, pre-culture duration, bacterial density (OD600), infection time, co-cultivation period, and antibiotic selection were systematically optimized. Putative transformants were screened by green fluorescence and verified by polymerase chain reaction (PCR). Results: Calli pre-cultured for 3 d were the most suitable recipient material. The optimal transformation conditions were an Agrobacterium suspension of OD600 = 0.2, infection for 5 min, and co-cultivation for 2 d; under these conditions, explant survival reached 72.22%. Two independent transgenic plantlets with stable integration of the enhanced green fluorescent protein gene were obtained and confirmed by PCR. Conclusions: This study provides an Agrobacterium-mediated transformation protocol for C. faberi and offers a technical basis for molecular breeding and gene function studies in this orchid.