Strategies for functional validation of genes involved in reproductive stages of orchids

Plants in the largest family of angiosperms, Orchidaceae, are diverse in both specialized pollination and ecological strategies and provide a rich source for investigating evolutionary relationships and developmental biology. However, studies in orchids have been hindered by several challenges that include low transformation efficiency and long regeneration time. To overcome such obstacles, we selected a symptomless cymbidium mosaic virus (CymMV) isolate for constructing virus-induced gene-silencing vectors. The feasibility of the virus vectors was first assessed with use of an orchid phytoene desaturase gene. The vector was able to induce gene silencing in orchids; however, because of the slow growth of orchids, the commonly used phytoene desaturase gene was not a good visual marker in orchids. We inserted a 150-nucleotide unique region of a B-class MADS-box family gene, PeMADS6, into pCymMV-pro60. The transcription level of PeMADS6 in inoculated Phalaenopsis plants was reduced by up to 73%, but no effect was observed for other MADS-box family genes. In contrast, in Phalaenopsis plants inoculated with CymMV transcripts containing 500 nucleotides of PeMADS6, a conserved region among MADS-box genes, the transcription level of PeMADS6 and the B- and C-class MADS-box genes was reduced by up to 97.8% as compared with plants inoculated with the vector alone. Flower morphology was affected in the MADS-box family gene-silenced plants as well. This in vivo experiment demonstrates an efficient way to study genes involved in the reproductive stage of plants with a long life cycle.