Establishment of Agrobacterium-mediated Genetic Transformation System for Cymbidium ensifolium ‘Shizhanghong’

Purpose To explore the genetic transformation system of Cymbidium ensifolium ‘Shizhanghong’ mediated by Agrobacterium.

Methods The rhizomes of C. ensifolium ‘Shizhanghong’ obtained from single pseudobulb propagation were used as experimental materials. Agrobacterium strain, concentration of acetosyringone, and mass concentration of antibiotics were screened. The main factors affecting the genetic transformation efficiency and regeneration efficiency of C. ensifolium ‘Shizhanghong’ mediated by Agrobacterium were studied.

Results The optimal strain for genetic transformation of C. ensifolium ‘Shizhanghong’ was EHA105, the optimal concentration of acetosyringone was 200 μmol/L, and the optimal mass concentration of hygromycin was 40 mg/L. To ensure a high transformation rate and high regeneration rate of C. ensifolium ‘Shizhanghong’, the optimal infection system was: after 10 days of pre-culture, the rhizomes were shaken in Agrobacterium infection solution (OD₆₀₀=0.4-0.6) for 20 minutes, and then co-cultured in the dark for three days. Through the screening of the optimal genetic transformation conditions, the transgenic positive plants of C. ensifolium ‘Shizhanghong’ were identified by green fluorescent protein, antibiotic screening, PCR and qRT-PCR detection. Finally, 17 transgenic positive plants of C. ensifolium ‘Shizhanghong’ were obtained from 180 rhizomes, with a transformation rate of 9.44%.

Conclusion This study establishes a genetic transformation system mediated by Agrobacterium for C. ensifolium ‘Shizhanghong’, laying the foundation for transgenic breeding of this orchid cultivar.