Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89197
標題: 生物技術創新扇葉文心蘭(Erycina pusilla)之研究
Studies on Developing Novel Erycina pusilla by Biotechnological Approach
作者: Chanon Lapjit
黃獻慶
關鍵字: 扇葉文心蘭
發光二極體
基因轉殖
化學誘變
Erycina pusilla
LEDs lights
gene transformation
chemical mutagenesis
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摘要: The purposes of this research were to manipulating the in vitro cultivation of Erycina pussila by LEDs illumination, to develop the novel, new flower color/type, and early blooming of Erycina pusilla via transformation of MADS14 (AP1-like) and CHRC genes, and to create new cultivars of Erycina pusilla by EMS and sodium azide mutagenesis and hybridization. The results indicated that compared with the fluorescent light and other LED treatments, RB+W+RGB LEDs significantly increased root and leaf number and plant high of in vitro plantlets of Erycina pusilla. RB+W+RB LEDs significantly increased the values of Fo, Fv, and Fv/Fm of in vitro plantlets after 25 weeks of cultivation, whereas significantly increased values of chlorophyll fluorescent parameters was found in the fluorescent light treatment after 50 weeks of cultivation. These results suggested RB+W+RGB LEDs and RB+W+RB LEDs could promote the quality of in vitro cultivation of Erycina pusilla. Three transformation vectors, p1305.3-CHRC, p1304-CHRC-IN, and p1301-AP1-MADS14 were transferred into the PLBs of the Erycina pusilla via Agrobacterium-mediated gene transformation. The regenerated plantlets were confirmed by PCR, RT-PCR and. Results indicated that the MADS14 gene was present in the genome of transformed Erycina pusilla plants and expressed its mRNA. Changes in the leaf and plant morphology, flower shape and color, blooming time, and multiple flower stalks were founded in the MADS14 transformed Erycina pusilla plants. This is the first report on the ectopic expression of MADS box gene in Erycina pusilla using an efficient and sophisticated gene transformation technology. In general, EMS treatments were more effective in reducing survival percentages and numbers of plant regeneration as compared to those of NaN3 treatments. Changes in profiles of flow cytometric histograms were found in several regenerated plants of EMS and NaN3 treated Erycina pulsilla PLB. In this study, 10 cultivar of Oncidiinae (♀) were crossed with Erycina pusilla (♂), in order to obtain novel Erycina pusilla. Several females tribute Oncidiinae crossed relatively easily with male Erycina pusilla but the reciprocal cross is rare. Hybrid seeds could be produced in Oncsa. Sweet Sugar x Erycina pusilla cross, and germinated under asymbiotic conditions. Changes in plant mophology and profiles of flow cytometric histograms were detected in vegetative leave of F1 plants.
本研究的目的為:1. 探討LED人工光源在扇葉文心蘭(Erycina pusilla)組織培養中的應用,2. 利用基因轉殖轉入MADS14 (AP1-like)新穎花型及花色、早開花之扇葉文心蘭,3. 經由EMS與疊氮化鈉 (sodium azide)等化學誘變和雜交的方式創新扇葉文心蘭的品種。 實驗結果顯示比較螢光燈管和不同LED燈,以紅藍光+白光+紅綠藍光 (RB+W+RGB) 的LED組合照射下,扇葉文心蘭的根和葉片的數量以及株高皆有顯著增加;而紅藍光+白光+紅藍光(RB+W+RGB)的LED組合中,在25週後Fo、Fv和Fv/Fm值皆顯著增加。而另外發現螢光燈管(fluorescent lamp)照射50週後,葉綠素螢光參數有明顯的增加。這些結果顯示紅藍光+白光+紅綠藍光(RB+W+RGB)以及紅藍光+白光+紅藍光(RB+W+RGB)的LED組合能夠在組織培養中提高扇葉文心蘭的品質。 三種農桿菌轉殖載體p1305.3-CHRC、p1304-CHRC-IN和p1301-AP1-MADS14經由農桿菌基因轉殖已經成功導入扇葉文心蘭擬原球體(PLBs)中,再生擬轉殖株經PCR和RT-PCR分析已確認MADS14基因整併到扇葉文心蘭的基因組中並且能經由轉錄作用表現其mRNA。在MADS14轉殖株系中發現到植株葉片形態、花型、花色、早開花以及多花梗等性狀的變化產生。這是首次在扇葉文心蘭中利用有效且精巧的基因轉殖技術,異源表現MADS box基因的報導。 EMS誘變處理相較於疊氮化鈉具有較低的存活率以及產生形態及花型變異的植株。由流式細胞儀分析的結果顯示經EMS和疊氮化鈉誘變的扇葉文心蘭植株,DNA發生改變。在組合親本雜交的試驗中,以10個文心蘭亞族的蘭花作為母本與扇葉文心蘭作為父本進行雜交以期產生新穎之扇葉文心蘭。研究結果發現文心蘭亞族的幾個蘭花與扇葉文心蘭具有親合性,雜交可產生果莢。其中Oncsa. Sweet Sugar (蜜糖) x Erycina pusilla之雜交種子可於無菌播種的環境下發芽,植株形態發生變異,以流式細胞儀分析F1子代的葉片,可偵測到DNA的改變。
URI: http://hdl.handle.net/11455/89197
其他識別: U0005-2506201516521100
文章公開時間: 2018-06-30
Appears in Collections:園藝學系

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