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Plant Physiology Preview Published on December 22, 2006; 10.1104/pp.106.092742
Received November 7, 2006 Strategies for Functional Validation of Genes Involved in Reproductive Stages of Orchids
Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 106, Taiwan; Department of Life Sciences, and Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan; Department of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan; Department of Horticulture, National Taiwan University, Taipei 106, Taiwan, Taipei 106, Taiwan * Corresponding author; email: hyeh{at}ntu.edu.tw.
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 (VIGS) vectors. The feasibility of the virus vectors was first assessed with use of an orchid phytoene desaturase gene (PDS). The vector was able to induce gene silencing in orchids; however, because of the slow growth of orchids, the commonly used PDS gene was not a good visual marker in orchids. We inserted a 150-nt 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 affect was observed for other MADS-box family genes. In contrast, in Phalaenopsis plants inoculated with CymMV transcripts containing 500 nt 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 is the first to demonstrate an efficient way to study genes involved in the reproductive stage of plants with a long life cycle.
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