S01 - Session P8 - Analysis of EIN2/EIN3 RNAi-mediated dual gene silencing in Oncidesa Gower Ramsey

Authors: Zhi Xing Lau, Yi-Yin Do, Pung-Ling Huang *

Oncidesa is the most exported cut flower from Taiwan. It is susceptible to ethylene, with especially the dislodgement of the pollinia cup during postharvest handling and transportation leading to ethylene production, which results in accelerated flower senescence. To prolong the longevity of Oncidesa cut flowers, positive regulators in the ethylene signaling pathway EIN2 and EIN3, were chosen to combine with the RNA interference (RNAi) technique and Agrobacterium -mediated transformationy. The EIN2 / EIN3 dual gene silencing construct was transformed into Oncidesa 'Gower Ramsey' and putative transgenic plants were analyzed in this study. The transformants obtained after antibiotic selection were confirmed by β-glucuronidase (GUS) histochemical staining analysis and the transformants with a positive staining result were further analysed by polymerase chain reaction (PCR) and Southern hybridization to determine transgene integrity. Among the phenotypes of the Oncidesa transformants measured, plant height, leaf length and root length of transgenic plants were shorter than those of non-transformants. Inverse PCR (IPCR) was utilized to analyze the insertion site of the T-DNA in the Oncidesa genome. Flanking sequences were obtained from ten transgenic lines, ranging from 11 bp to 133 bp in length and were characterized into four groups. For one of them it was found that the T-DNA was inserted into the 5'-untranslated region (5'-UTR) of the carbon catabolite repressor protein 4 homolog 1 ( CCR4-1 ) gene.