The detection of siRNA was carried out as described previously using digoxogenin-labeled rose DFR cDNA as a probe, as previously described (Goto et al. 2003).
The procedures involving binary vectors for rose transformation were also described previously (Fukuchi-Mizutani et al. 2003).
In brief, pBin19 and pBinPLUS (van Engelen et al. 1995), which contain neomycin phosphotransferase II (NPTII) as the selection marker, were used to construct a binary vector, and the enhanced cauliflower mosaic virus (CaMV) 35S promoter derived from pE2113 (Mitsuhara et al. 1996) was used to regulate the transgenes.
Torenia anthocyanin 5-acyltransferase cDNA (database accession No. AB332099) was obtained from its petal cDNA library (Suzuki 2000) as described previously (Yonekura-Sakakibara et al. 2000).