Development and application of the TRV-induced gene-silencing system in different Rhododendron species

Rhododendron is one of the top ten traditional flowers in China, with high ornamental and medicinal values. However, molecular research on this species has been impeded by the lack of efficient molecular genetic techniques. Virus-induced gene silencing (VIGS) technology is an effective tool for analyzing gene functions, which has been successfully applied to many plant species. But there have been no reports of VIGS system for Rhododendron. In this study, tobacco rattle virus (TRV) was chosen to infect Rhododendron micranthum using phytoene desaturase (PDS) as the reporter gene. After the inoculation of pTRV2-RmPDS by leaf injection, photobleaching phenomena appeared in the newly developed leaves and the RT-qPCR assay demonstrated that RmPDS was successfully silenced. Then three parameters including the developmental stage, the Agrobacterium concentration and the inoculation temperature were examined to optimize the system. The silencing efficiency was increased from 2.4 to 11.4% and the optimized conditions were as follows: the developmental stage of the two true-leaf stage, the adjustment of the inoculation solution to a final OD₆₀₀ = 1.5 and the inoculation temperature of 18 ℃. To further validate the system, the most optimal combination was used to infect other six rhododendron genotypes. R. mucronulatum, R. ovatum, R. × pulchrum, R. simsii and R. yedoense displayed the silenced phenotype of PDS as expected. We successfully established TRV-mediated VIGS technology in Rhododendron which could evaluate and characterize the function of plant genes without the need for cumbersome tissue culture.