Optimization of Virus-induced Phytoene Desaturase (PDS) gene silencing using Tobacco Rattle Virus in Coleus forskohlii

Virus-induced gene silencing (VIGS) is an effective reverse genetics method used to study the gene function by analysis of plants at phenotype and molecular levels. Here, the standardization of the protocol for VIGS through Agrobacterium-mediated transformation was successfully achieved. Stem cuttings with roots were established in Coleus forskohlii. Phytoene desaturase gene from Coleus forskohlii (CfPDS) was isolated and cloned in TRV2 vector to construct CfPDS:TRV2. CfPDS:TRV2 construct was transformed in Agrobacterium tumefaciens. Agro-infiltration was carried out using leaf infiltration, agrodrench and submerged methods. Submerged method gave prominent results as compared to others. The expression of CfPDS gene was significantly decreased in plants agro-infiltrated with CfPDS:TRV2 construct resulting in photobleached areas of the affected parts of leaves. CfPDS:TRV2 treated plants showed lower chlorophyll a concentration and production of reactive oxygen species (ROS) as compared to TRV1 + 2 treated control plants. This study resulted in the establishment of VIGS protocol for Coleus forskohlii that can be further used to engineer the genes involved in the secondary metabolite pathway.