Optimization of Agrobacterium-mediated transient gene expression and endogenous gene silencing in Piper colubrinum Link. by vacuum infiltration

Abstract

The present study describes the successful development of vacuum infiltration method in the oomycete-resistant wild Piper sp., Piper colubrinum, as a rapid transient method for expression of GUS (β-Glucuronidase) reporter gene and introduction of hairpin vector for endogenous gene silencing. The GUS reporter gene construct pCAMBIA 1305.2 was used as a positive control to test the efficiency of vacuum infiltration strategy. Agrobacteria (EHA 105) harbouring GUS binary vector were vacuum-infiltrated into young detached in vitro leaf explants, which showed detectable GUS gene activity within 4 days of infiltration. This paper also reports for the first time the application of transient gene silencing in P. colubrinum by the delivery of in vitro synthesized hairpin vector construct (pHELLSGATE) containing endogenous serine threonine protein kinase (STPK) gene homologue into in vitro shoots. Introduction of hairpin vectors for the STPK gene into in vitro plantlets by vacuum infiltration resulted in significant reduction in transcript accumulation of the endogenous gene. The results indicate that transient gene silencing could be used as a rapid, preliminary high-throughput tool for P. colubrinum functional genomics.