|Reid, Michael - DEPT PLANT SCI, UC DAVIS|
|Chen, Jen-Chih - DEPT PLANT SCI, UC DAVIS|
Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: December 11, 2008
Publication Date: January 1, 2009
Citation: Reid, M.S., Chen, J., Jiang, C. (2009) VIRUS-INDUCED GENE SILENCING FOR FUNCTIONAL CHARACTERIZATION OF GENES IN PETUNIA. In Petunia: Evolutionary, Developmental and Physiological Genetics. T. Gerats ed. Springer, New York, pp381-394. Technical Abstract: Although functional analysis of genes can be readily carried out in Petunia using standard transformation/regeneration techniques, this process is time- and labor-consuming. High throughput analysis of gene knockout has been made possible by the use of Virus-Induced Gene Silencing; fragments of target plant genes are included in the genome of a viral vector, and the plant silences them as part of its viral defense mechanism. In petunia, we use a modified tobacco rattle virus (TRV) vector for VIGS. The TRV RNA2 vector includes a multiple cloning site into which we can insert fragments of target genes. Infection typically results in chimeric plants, and it is therefore desirable to have a reporter that can show where target genes have been silenced. Inserting a fragment of the gene encoding phytoene desaturase (PDS) results in a photo-bleaching phenotype where silencing occurs in the leaves. Inserting a fragment of the gene encoding chalcone synthase (CHS) in the viral vector allows us to visualize silencing in floral tissues of purple-flowered petunia cultivars as white patches, sectors or even entire corollas. We have shown that the VIGS system can silence as many as five independent genes at one time. We describe here the methods that we have found to be efficient and effective for VIGS in petunia, and describe some results obtained by silencing a range of genes, including some transcription factors.