RNA interference (RNAi) is a posttranscriptional gene silencing phenomenon induced by double-stranded RNA. It has been widely used as a knockdown technology to analyze gene function in many organisms. In tomato, RNAi technology has widely been used as a reverse genetic tool for functional genomics study. Generally, RNAi is often achieved through transgenes producing hairpin RNA molecules. RNAi lines have the advantage with respect to more modern CRISPR/Cas9 mutants of different levels of downregulation of target gene, and allow the characterization of life-essential genes that cannot be knocked out without killing the organism. Also, RNAi allows to suppress gene expression in multigene families in a regulated manner. In this chapter, an efficient approach to create RNAi stable knockdown-transformed tomato lines is reported. In order, it describes the choice of the target silencing fragment, a highly efficient cloning strategy for the hairpin RNA construct production, a relatively easy procedure to transform and regenerate tomato plants using Agrobacterium tumefaciens and a methodology to test the goodness of the transformation procedure.
Keywords: Agrobacterium-mediated transformation; Gateway cloning; RNA interference; Silencing; Tomato.