Osmotic sucrose enhancement of single-cell embryogenesis and transformation efficiency in <Emphasis Type="Italic">Oncidium</Emphasis> |
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Authors: | Shu-Hua?Li Chang-Sheng?Kuoh Yau-Huang?Chen Hong-Hwa?Chen Email author" target="_blank">Wen-Huei?ChenEmail author |
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Institution: | (1) Institute of Biotechnology, National Cheng Kung University, Tainan, 701, Taiwan;(2) Department of Biological Science, National Cheng Kung University, Tainan, 701, Taiwan;(3) Plant Propagation Center, Taiwan Sugar Research Institute, Tainan, 702, Taiwan;(4) Present address: Tainan District of Agricultural Research and Extention Station, Tainan, 712, Taiwan;(5) Institute of Botany, Academia Sinica, Taipei , 115, Taiwan;(6) Department of Life Sciences, National University of Kaohsiung, Kaohsiung, 811, Taiwan |
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Abstract: | Traditional breeding processes to genetically modify the long reproductive cycle and slow seed maturation of orchids have limits. We developed a more efficient protocol using particle bombardment to produce transgenic plants of Oncidium Sharry Baby OM8 (Orchidaceae). Pretreating protocorm-like bodies (PLBs) with 0.5 M sucrose for 2 h increased single-cell embryogenesis 3- to 4-fold; however, shoot formation was suppressed. In addition, new PLBs were regenerated from the entire sucrose-pretreated PLBs, whereas in untreated PLBs, this occurred only from the bases. Pretreated PLBs were bombarded with pSPFLP containing genes encoding a sweet pepper ferredoxin-like protein (pflp), hygromycin phosphotransferase (hpt) and -glucuronidase (GUS) driven by the cauliflower mosaic virus 35S promoter. Pretreated PLBs showed a 14.8-fold increase in GUS expression over the untreated PLBs 40days after bombardment. The presence of pflp and hpt transgenes in the 40 putatively stably transformed lines that produced 113 clones was confirmed by PCR analysis. Six lines (eight clones) were positive for both pflp and hpt transgenes. In addition, clones derived from these lines were either all positive or all negative for the two transgenes, which suggests homogeneity in pretreated PLBs with more single-cell embryogenesis. Thus, sucrose pretreatment enhanced the regeneration of PLBs, single-cell embryogenesis and efficiency of transformation. |
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Keywords: | gene transformation Oncidium particle bombardment protocorm-like body (PLB) single-cell embryogenesis |
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