RNAi Mediated Down-Regulation of PDS Gene Expression in Sugarcane (Saccharum), a Highly Polyploid Crop

Sugarcane is a crop with great potential for metabolic engineering, but progress has been limited by highly efficient transgene silencing. The potential exists to utilize efficient gene silencing in molecular improvement through down-regulation of sugarcane genes. However, sugarcane is highly polyploid and heterozygous, which might complicate efforts to employ transgene-mediated silencing of endogenous genes. To explore this issue, we tested in sugarcane a construct designed for hairpin-mediated silencing of the phytoene desaturase (PDS) gene in sugarcane. The hairpin construct was designed based on the sugarcane PDS EST collection containing multiple alleles, to down-regulate the suite of PDS alleles. Three out of four plants transformed using the PDS hairpin construct showed detectable hairpin transcripts when analyzed by northern analysis, and displayed the photo-bleaching phenotype characteristic of PDS knockout lines. There was near-complete reduction in the levels of endogenous PDS transcripts in leaf tissue, indicating efficient hairpin-mediated down-regulation despite the highly polyploid and heterozygous sugarcane genome. Hairpin-mediated gene silencing should therefore be a powerful tool for the molecular improvement of this important crop.