臺灣博碩士論文加值系統

蝴蝶蘭是蘭科植物中最重要的經濟栽培作物，可供商業切花和盆花的生產，行政院農業委員會業已將其選定為台灣四大外銷旗艦農產品之一。在台灣，無論是蝴蝶蘭生產或品種改良等研發，應具有可觀的預期經濟利益。然而，蘭花通常擁有較長之幼年期與生殖周期，此特性某種程度局限了傳統雜交育種的改良，現代遺傳工程則是一條可行的品種創育途徑。然而，體細胞變異、試管內選拔和遺傳工程等作物遺傳改良之技術，皆需要有高效率且穩定地經由癒合組織再生植株的體系為基礎。本論文研究之目的擬建立蝴蝶蘭經由癒合組織再生植株的高效率體系，並架構蝴蝶蘭基因轉殖代工平台，期能整合國內蝴蝶蘭分子育種體系，以因應未來蝴蝶蘭品種多樣化之育種需求，期能奠定我國蝴蝶蘭外銷產業堅實的基礎。

Phalaenopsis orchid is one of the most important orchids grown for commercial production of cut flowers and potted plants. It has been chosen by the Council of Agriculture, Executive Yuan, as one of the four flagship speciality products for export. There is substantial interest in the production and improvement of these commercially valuable plants in Taiwan. However, orchids usually have long juvenile periods and reproductive cycles, which restrict genetic improvement using traditional sexual hybridization. Modern genetic engineering techniques provide an effective alternative. A callus-mediated plant regeneration protocol is a critical requirement as it allows exploitation of in vitro selection, somaclonal variation and engineering techniques aimed at genetic improvement of plants. Although several micropropagation methods have been developed for phalaenopsis using shoot tips, leaf segments, root tips and stalk cuttings, establishment of a proliferating callus culture and regeneration from callus is limited to phalaenopsis. The objectives of the project are to develop an efficient plant regeneration system from callus and to establish a platform of gene transformation for phalaenopsis breeding purpose, hoping to lay a solid foundation for export-oriented businesses.

目 錄

口試委員會審定書……………………………………………………………………… i
序言………………………………………………………………… iii
中文摘要…………………………………………………………… iv
英文摘要……………………………………………………………… v
第一章 緒論………………………………………………………… 001
1.1 重要蝴蝶蘭消費市場規模的演變 …………………………… 001
1.2 臺灣蝴蝶蘭產業的現況 ……………………………………… 002
1.3 臺灣蝴蝶蘭產業永續發展的技術需求 ……………………… 005
第二章 前人研究 ………………………………………………… 007
2.1 蝴蝶蘭的基因轉殖系統……………………………………… 007
2.1.1 蝴蝶蘭的農桿菌媒介基因轉殖系統……………………… 008
2.1.2 蝴蝶蘭的基因槍轉殖系統………………………………… 012
2.2 蝴蝶蘭基因轉殖的篩選與報導系統 ……………………… 014
2.2.1 植物基因轉殖常用的篩選基因與試劑 ………………… 014
2.2.2 蝴蝶蘭基因轉殖採用的篩選基因與試劑………………… 016
2.2.3 蝴蝶蘭基因轉殖採用的報導基因系統…………………… 018
2.3 蝴蝶蘭基因轉殖體系採用的組織培養之植株再生系統…… 020
2.3.1 蝴蝶蘭基因轉殖體系採用轉殖處理植物材料的考量…… 020
2.3.2 蝴蝶蘭組織培養植株再生體系與基因轉殖發展需求…… 021
2.4 蝴蝶蘭基因轉殖體系的應用現況與策略……………………023
第三章 蝴蝶蘭癒合組織再生植株體系的建立………………… 024
3.1 摘要…………………………………………………………… 026
3.2 前言…………………………………………………………… 025
3.3 材料與方法…………………………………………………… 026
3.3.1 試驗材料與培養環境……………………………………… 026
3.3.2 蝴蝶蘭癒合組織生長曲線試驗…………………………… 026
3.3.3 蝴蝶蘭癒合組織分化擬原球莖體試驗…………………… 026
3.3.4 蝴蝶蘭癒合組織分化之擬原球莖體其再生植株之生長試驗
…………………………………………………………… 027
3.3.5 培養基鹽類組成對蝴蝶蘭幼苗生長之影響……………… 027
3.3.6 蝴蝶蘭癒合組織分化擬原球莖體、再生植株之形態觀察
………………………………………………………… 027
3.3.7 生長調節劑對朵麗蝶蘭癒合組織增殖之影響………….. 027
3.3.8 糖類和濃度對朵麗蝶蘭癒合組織分化擬原球莖體之影響
...………………………………………………………… 028
3.3.9 蔗糖和硝酸銨濃度對朵麗蝶蘭再生植株生長之影響…… 028
3.3.10 朵麗蝶蘭癒合組織分化擬原球莖體、再生植株之形態觀察
………………………………………………………… 028
3.4 結果與討論…………………………………………………… 028
3.4.1 建立蝴蝶蘭癒合組織之植株再生體系…………………… 028
3.4.1.1 蝴蝶蘭癒合組織生長曲線試驗………………………... 028
3.4.1.2 蝴蝶蘭癒合組織分化擬原球莖體試驗………………… 029
3.4.1.3 培養基鹽類、糖類對蝴蝶蘭擬原球莖體再生植株之影響
...……………………………………………………… 032
3.4.1.4培養基鹽類組成對蝴蝶蘭幼苗生長之影響…………… 032
3.4.1.5 蝴蝶蘭癒合組織分化擬原球莖體、再生植株之形態觀察
…………………………………………………….… 032
3.4.2 建立朵麗蝶蘭癒合組織之植株再生系統………………... 033
3.4.2.1 生長調節劑對朵麗蝶蘭癒合組織增殖之影響………. 033
3.4.2.2糖類和濃度對朵麗蝶蘭癒合組織分化擬原球莖體之影響
……………………………………………………… 033
3.4.2.3蔗糖和硝酸銨濃度對朵麗蝶蘭癒合組織再生幼苗生長的影響………………………………………………………… 035
3.4.2.4朵麗蝶蘭癒合組織分化擬原球莖體、再生植株之形態觀察
…...…………………………………………………… 035
第四章 蝴蝶蘭農桿菌媒介基因轉殖系統的建立………………….. 060
4.1 摘要…………………………………………………………060
4.2 前言…………………………………………………………062
4.3 材料與方法…………………………………………………… 062
4.3.1轉殖標的植物材料與培養環境…………………………. 062
4.3.2 蝴蝶蘭癒合組織對篩選試劑的天然抗性…………………. 062
4.3.2.1 白花蝴蝶蘭癒合組織對遺傳黴素和西弗士林的天然抗性062
4.3.2.2 白花蝴蝶蘭癒合組織對潮黴素的天然抗性…………….. 063
4.3.2.3 白花紅心朵麗蝶蘭癒合組織對遺傳黴素和潮黴素的天然抗性063
4.3.3農桿菌媒介基因轉殖…………………………………… 064
4.3.3.1 農桿菌品系與質體……………..……………………… 064
4.3.3.2 共培養的基本流程…………………..……….………… 065
4.3.3.3 農桿菌菌濃度與共培養時間對轉殖效率的影響…… 066
4.3.3.4 乙醯丁香酮濃度對轉殖效率的影響………………… 066
4.3.3.5 癒合組織培養天數對轉殖效率的影響……………… 066
4.3.3.6 轉殖癒合組織重複轉殖的可行性與效率.…………… 067
4.3.3.7朵麗蝶蘭白花紅心品種的轉殖試驗………………… 067
4.3.3.8 篩選與植株再生…………………………………… 067
4.3.4 轉殖事件的評析………………………………………… 068
4.3.4.1 GUS活性的組織化學染色法………………………… 068
4.3.4.2 GFP螢光鏡檢………………………………………… 068
4.3.4.3 植物基因組DNA的抽取…….……………………… 068
4.3.4.4 聚合酶連鎖反應……………………………… 069
4.3.4.5 植物基因組DNA酵素酶切與純化……………… 069
4.3.4.6 植物總量RNA的抽取……………………………… 070
4.3.4.7 反轉錄聚合酶連鎖反應……………………………… 070
4.3.4.8轉殖體之核內多倍體細胞核分布的檢測……… 071
4.3.4.9 間接式酵聯抗體法………………………… 071
4.3.4.10南方氏核酸雜交法……………………………… 072
4.4 結果與討論…………….………………………………… 072
4.4.1蝴蝶蘭癒合組織對篩選試劑的天然抗性…………… 072
4.4.1.1 白花蝴蝶蘭癒合組織對遺傳黴素和西弗士林的天然抗性... 072
4.4.1.2白花蝴蝶蘭癒合組織對潮黴素的天然抗性……… 074
4.4.1.3白花紅心朵麗蝶蘭癒合組織對遺傳黴素和潮黴素的天然抗性 074
4.4.2 蝴蝶蘭農桿菌媒介基因轉殖系統的建構………… 075
4.4.2.1農桿菌菌濃度與共培養時間對轉殖效率的影響............. 076
4.4.2.2乙醯丁香酮濃度對轉殖效率的影響........................... 076
4.4.2.3 癒合組織培養天數對轉殖效率的影響........................ 077
4.4.2.4轉殖癒合組織重複轉殖的可行性與效率.....................077
4.4.2.5白花紅心朵麗蝶蘭的轉殖試驗……….........................078
4.4.3蝴蝶蘭農桿菌媒介基因轉殖事件的評析………079
4.4.3.1聚合酶連鎖反應分析……………………………079
4.4.3.2基因組DNA經限制酶酶切再進行聚合酶連鎖反應的分析...081
4.4.3.3 總量RNA反轉錄聚合酶連鎖反應分析.............................082
4.4.3.4核內再複製現象與轉殖系聚合酶連鎖反應分析結果的相關性083
4.4.3.5 間接式酵聯抗體法分析......................................085
4.4.3.6南方氏核酸雜交法分析..................................085
第五章 應用綠色螢光蛋白報導基因探討蝴蝶蘭癒合組織基因槍轉殖暫時性表達之最佳物理參數……………………………………………….. 121
5.1 摘要…………………………………………………………… 121
5.2 前言…………………………………………………………… 122
5.3 材料與方法…………………………………………………… 123
5.3.1 植物材料之培養與製備……………………………………. 123
5.3.2 質體DNA與微粒子製備…………………………………… 123
5.3.3 基因槍轉殖處理…………………………………………... 124
5.3.3.1 參試蝴蝶蘭癒合組織最適之基因槍轉殖物理參數…… 124
5.3.3.2 基因槍轉殖蝴蝶蘭癒合組織表達GFP之時程試驗…… 124
5.3.4 GFP螢光鏡檢………………………………………………. 124
5.4 結果與討論…………………………………………………… 125
5.4.1 最適化基因槍轉殖蝴蝶蘭癒合組織之物理參數……….. 125
5.4.2 基因槍轉殖蝴蝶蘭癒合組織表達GFP之時程試驗……… 125
5.4.3 應用GFP報導系統提升蝴蝶蘭轉殖體系建構之評估….. 126
第六章 葡萄糖苷酸酶活性組織化學染色法分析蝴蝶蘭轉殖事件效果之改進………………………………………………..………….…. 132
6.1 摘要…………………………………………………………… 132
6.2 前言…………………………………………………………… 133
6.3 材料與方法…………………………………………………… 134
6.3.1 植物材料……………………………………………...…… 134
6.3.2 GUS活性組織化學染色法分析蝴蝶蘭轉殖癒合組織的改進試驗…………………………………………………………… 135
6.3.3 GUS活性組織化學染色法分析蝴蝶蘭轉殖葉圓片的改進試驗
…………………………………………………………… 135
6.4 結果與討論…………………………………………………… 136
6.4.1 GUS活性組織化學染色法分析蝴蝶蘭轉殖癒合組織的改進試驗…………………………………………………………… 136
6.4.2 GUS活性組織化學染色法分析蝴蝶蘭轉殖葉圓片的改進試驗
…………………………………………………………… 137
6.4.3 GUS活性組織化學染色改進法的評估…………………… 138
第七章 轉殖蝴蝶蘭雙報導基因表達之檢測……………………….. 149
7.1 摘要…………………………………………………………… 149
7.2 前言…………………………………………………………… 150
7.3 材料與方法…………………………………………………… 152
7.3.1 蝴蝶蘭ATK32轉殖系癒合組織、原球莖體與擬轉殖株雙報導基因gus與gfp表達之觀察………………………………………… 152
7.3.2 蝴蝶蘭ATK32轉殖系癒合組織與擬轉殖株原生質體GFP螢光表現之檢測……………………………………………………………. 153
7.3.2.1 癒合組織與其原生質體之GFP螢光表現觀察………… 153
7.3.2.2 原生質體之流式細胞儀分析GFP螢光表現………… 153
7.3.3 蝴蝶蘭ATK32轉殖系癒合組織之體胚發生過程觀察…… 153
7.4 結果與討論……………………………………………………… 154
7.4.1 蝴蝶蘭ATK32轉殖系癒合組織、原球莖體與擬轉殖株雙報導基因gus與gfp表達之觀察………………………………………… 154
7.4.2 蝴蝶蘭ATK32轉殖系癒合組織與擬轉殖株原生質體GFP螢光表現之檢測……………………………………………………………. 156
7.4.2.1 GFP螢光表現觀察……………………………………… 156
7.4.2.1.1 癒合組織與其原生質體之GFP螢光表現觀察……… 156
7.4.2.1.2 擬轉殖株原生質體之GFP螢光表現觀察………… 156
7.4.2.2 原生質體之流式細胞儀分析GFP螢光表現………… 157
7.4.2.2.1癒合組織原生質體之流式細胞儀分析GFP螢光表現157
7.4.2.2.2擬轉殖株原生質體之流式細胞儀分析GFP螢光表現
………………………………………………………… 159
7.4.3 蝴蝶蘭ATK32轉殖系癒合組織之體胚發生過程觀察…… 160
第八章 結論…………………………………………… 176
參考文獻………………………………………………178
附錄……………………………. 192



圖目錄
圖3.1蝴蝶蘭癒合組織之累計生長曲線圖 …………………………………050
圖3.2蝴蝶蘭癒合組織之相對增長曲線圖 ………………………………………050
圖3.3不同鹽類組成對蝴蝶蘭幼苗發育之影響 …………………………………051
圖3.4白花蝴蝶蘭癒合組織經分化擬原球莖體之植株再生過程…………………052
圖3.5. 蝴蝶蘭（Phalaenopsis Taisuco Kaaladian）癒合組織之體胚發生過程… 053
圖3.6生長調節劑對朵麗蝶蘭癒合組織增殖之影響 ……………………………054
圖3.7糖類和濃度對朵麗蝶蘭癒合組織形態分化之影響（I） …………………054
圖3.8糖類和濃度對朵麗蝶蘭癒合組織形態分化之影響（II）……………..……055
圖3.9於含不同糖類和濃度培養基上分化之朵麗蝶蘭擬原球莖體，於HS培養發育之情形 ………………………………………………………………056
圖3.10蔗糖和硝酸銨濃度對朵麗蝶蘭癒合組織再生幼苗生長的影響（I） ……057
圖3.11蔗糖和硝酸銨濃度對朵麗蝶蘭癒合組織再生幼苗生長的影響（II） ……058
圖 3.12白花紅心朵麗蝶蘭癒合組織經分化擬原球莖體之植株再生過程……059
圖4.1 參試質體的構築與引子位置圖…………………………………………100
圖4.2遺傳黴素和西弗士林對白花蝴蝶蘭癒合組織於不同測試條件下存活的影響………………………………………………………………………101
圖4.3 潮黴素對白花蝴蝶蘭癒合組織存活的影響……………………………101
圖4.4 遺傳黴素和潮黴素對白花紅心朵麗蝶蘭癒合組織存活的影響………104
圖4.5 農桿菌媒介基因轉殖蝴蝶蘭表達報導基因的情形………………102
圖4.6 蝴蝶蘭轉殖系ATK019再生植株的聚合酶鏈鎖反應分析結果………103
圖4.7 蝴蝶蘭ATL001轉殖系再生植株進行聚合酶鏈鎖反應分析的結果…104
圖4.8 蝴蝶蘭ATL003轉殖系再生植株進行聚合酶鏈鎖反應分析的結果…105
圖4.9 蝴蝶蘭ATL006轉殖系再生植株進行聚合酶鏈鎖反應分析的結果…106
圖4.10 蝴蝶蘭ATK016轉殖系再生植株進行聚合酶鏈鎖反應分析的結果…107
圖4.11 蝴蝶蘭ATK023轉殖系再生植株進行聚合酶鏈鎖反應分析的結果…108
圖4.12 蝴蝶蘭ATK032轉殖系再生植株進行聚合酶鏈鎖反應分析的結果（I）109
圖4.13 蝴蝶蘭ATK032轉殖系再生植株進行聚合酶鏈鎖反應分析的結果（II）110
圖4.14 蝴蝶蘭ATK023轉殖系再生植株基因組DNA限制酶酶切後進行聚合酶鏈鎖反應分析的結果……………………………………………………….111
圖4.15 蝴蝶蘭ATK032轉殖系再生植株基因組DNA限制酶酶切後進行聚合酶鏈鎖反應分析的結果…………………………………………………….112
圖4.16 蝴蝶蘭ATK023轉殖系植株進行反轉錄聚合酶鏈鎖反應分析的結果….113
圖4.17 蝴蝶蘭ATK032轉殖系植株進行反轉錄聚合酶鏈鎖反應分析的結果(I)114
圖4.18 蝴蝶蘭ATK032轉殖系植株進行反轉錄聚合酶鏈鎖反應分析的結果(II)115
圖4.19 蝴蝶蘭ATK023和ATK032轉殖系癒合組織進行聚合酶鏈鎖反應的結果………………………………………..……………………………………116
圖4.20 蝴蝶蘭ATK023和ATK032轉殖系參試質體進行聚合酶鏈鎖反應的結果………………………….……………..……………………………………117
圖4.21白花蝴蝶蘭（Phalaenopsis Taisuco Kaaladian）ATK023轉殖系癒合組織與擬轉殖株葉片細胞核的DNA含量與核內多倍體分布之分析……………………………….………………………………………….117
圖4.22 蝴蝶蘭ATK032轉殖系再生植株以間接式酵聯抗體法檢測惠蘭嵌紋病毒和齒舌蘭輪點病毒鞘蛋白的結果………….…………………………….118
圖4.23 蝴蝶蘭ATK019轉殖系再生擬轉殖植株的南方氏核酸雜交法分析結果119
圖4.24 蝴蝶蘭ATK016轉殖系再生擬轉殖植株的南方氏核酸雜交法分析結果120
圖5.1 基因槍轉殖之蝴蝶蘭癒合組織表達GFP的時程變化曲線 ……………129
圖5.2 基因槍轉殖之蝴蝶蘭癒合組織GFP表達隨時間變化的情形 …………130
圖5.3 蝴蝶蘭基因槍暫時轉殖細胞之GFP表達量隨時間改變的情形 ………131
圖6.1 蝴蝶蘭轉殖癒合組織以不同GUS活性組織化學染色法處理6小時內的呈色表現…………………………………………………………………………142
圖6.2 蝴蝶蘭轉殖癒合組織以不同GUS活性組織化學染色法處理48小時內的呈色表現………………………………………………………………………143
圖6.3蝴蝶蘭轉殖植株葉圓片以不同GUS活性組織化學染色法處理6小時後的呈色表現………………………………………………………………144
圖6.4蝴蝶蘭轉殖植株葉圓片以1％ Triton X-100磷酸緩衝溶液進行不同時間的前處理，再以GUS染色溶液處理24小時的呈色結果…………145
圖6.5蝴蝶蘭轉殖植株葉圓片以2％ Triton X-100磷酸緩衝液進行不同時間的前處理，再以GUS染色溶液處理24小時的呈色結果………………………145
圖6.6蝴蝶蘭轉殖植株葉圓片以Clorox（20％）進行不同時間的前處理，再以GUS染色溶液處理24小時的呈色結果………………………………146
圖6.7蝴蝶蘭轉殖植株葉圓片以乙醇（95％）進行不同時間的前處理，再以GUS染色溶液處理24小時的呈色結果…………………………………………146
圖6.8蝴蝶蘭轉殖植株葉圓片以甲醇進行不同時間的前處理，再以GUS染色溶液處理24小時的呈色結果…………………………………………147
圖6.9未經Triton X-100磷酸緩衝液前處理的轉殖蝴蝶蘭癒合組織，呈色不佳（左）。但該癒合組織於染色24小時後，重新先經1％ Triton X-100前處理並以不抽真空處理的方式再染色，其呈色情形（右，再染色後10小時拍照）可如Triton X-100前處理者相似。…………………………………147
圖6.10蝴蝶蘭轉殖植株GUS活性組織化學染色之表現，隨著擬原球莖體發育成植株，GUS之呈色反應亦下降，尤其是在葉片上不易見到藍色表現…148
圖7.1蝴蝶蘭（Phalaenopsis Taisuco Kaaladian）ATK32轉殖癒合組織再生擬原球莖體之雙報導基因gfp與gus表達（I）………………………………163
圖7.2蝴蝶蘭（Phalaenopsis Taisuco Kaaladian）ATK32轉殖癒合組織再生擬原球莖體之雙報導基因gfp與gus表達（II）………………………………164
圖7.3蝴蝶蘭（Phalaenopsis Taisuco Kaaladian）ATK32轉殖癒合組織再生擬原球莖體之雙報導基因gfp與gus表達（III）………………………………165
圖7.4蝴蝶蘭（Phalaenopsis Taisuco Kaaladian）ATK32轉殖癒合組織再生植株之gfp報導基因表達。……………………………………………………166
圖7.5蝴蝶蘭ATK32轉殖癒合組織與其原生質體之GFP螢光表現觀察………169
圖7.6 蝴蝶蘭ATK32轉殖細胞系之GFP表達點陣圖 ……………………………170
圖7.7 蝴蝶蘭ATK32轉殖細胞系之GFP表達統計圖 ……………………………171
圖7.8蝴蝶蘭ATK32擬轉殖株原生質體之GFP螢光表現觀察……………………172
圖7.9 蝴蝶蘭ATK32擬轉殖株之GFP表達點陣圖 ………………………………173
圖7.10 蝴蝶蘭ATK32轉殖細胞系之GFP表達統計圖……………………………174
圖7.11 由蝴蝶蘭ATK32轉殖系細胞之GFP螢光表現觀察體胚發生過程………175

表目錄
表3.1 蝴蝶蘭不同初始繼代細胞量對後續增殖之影響……………………………036
表3.2三種雙醣種類和濃度以及培養方式對蝴蝶蘭癒合組織再生培養之鮮重、葉綠素及類胡蘿蔔素之影響……………………………………………………036
表3.3變方分析三種雙醣種類和濃度以及培養方式對蝴蝶蘭癒合組織再生培養之鮮重、葉綠素及類胡蘿蔔素之影響…………………………………………037
表3.4三種雙醣種類和濃度以及培養方式對蝴蝶蘭癒合組織再生培養之鮮重與擬原球莖體形成之影響（I）……………………………………………………038
表3.5三種雙醣種類和濃度以及培養方式對蝴蝶蘭癒合組織再生培養之鮮重與擬原球莖體形成之影響（II）……………………………………………………039
表3.6變方分析三種雙醣種類和濃度以及培養方式對蝴蝶蘭癒合組織再生培養之鮮重與擬原球莖體形成之影響……………………………………………040
表3.7不同培養基鹽類、雙醣種類和濃度對蝴蝶蘭擬原球莖體之植株再生影響041
表3.8變方分析培養基鹽類、雙醣種類和濃度對蝴蝶蘭擬原球莖體發育植株之影響
…………………………………………………………………………041
表3.9培養基鹽類組成對蝴蝶蘭幼苗生長之影響………………………………042
表3.10生長調節劑對朵麗蝶蘭癒合組織增殖之影響……………………………042
表3.11朵麗蝶蘭癒合組織培養於含三種雙醣種類和濃度的分化培養基之生長情形
…………………………………………………………………………043
表3.12變方分析三種雙醣種類和濃度對朵麗蝶蘭癒合組織分化之影響………044
表3.13三種雙醣種類和濃度對朵麗蝶蘭癒合組織之擬原球莖體分化及發育之影響
…………………………………………………………………………045
表3.14變方分析三種雙醣種類和濃度對朵麗蝶蘭癒合組織之擬原球莖體分化及發育之影響…………………………………………………………………046
表3.15來自不同糖類和濃度培養基之朵麗蝶蘭擬原球莖體，於HS培養基幼苗發育之情形…………………………………………………………………047
表3.16蔗糖和硝酸銨濃度對朵麗蝶蘭癒合組織再生幼苗生長的影響（I）……047
表3.17蔗糖和硝酸銨濃度對朵麗蝶蘭癒合組織再生幼苗生長的影響（II）……048
表3.18變方分析蔗糖和硝酸銨濃度對朵麗蝶蘭癒合組織再生幼苗生長的影響
…………………………………………………………………………048
表3.19變變方分析蔗糖和硝酸銨濃度對朵麗蝶蘭癒合組織再生幼苗根葉比值的影響………………………………………………………………………049
表4.1 遺傳黴素對白花蝴蝶蘭癒合組織於不同測試條件下存活的影響…………087
表4.2 遺傳黴素和西弗士林對白花蝴蝶蘭癒合組織於不同測試條件下存活影響的變方分析………………………………………………………………088
表4.3潮黴素對白花蝴蝶蘭癒合組織存活的影響（I）…………………088
表4.4潮黴素對白花蝴蝶蘭癒合組織存活的影響（II）………………089
表4.5遺傳黴素對白花紅心朵麗蝶蘭癒合組織存活的影響（I）……089
表4.6遺傳黴素對白花紅心朵麗蝶蘭癒合組織存活的影響（II）………090
表4.7潮黴素對白花紅心朵麗蝶蘭癒合組織存活的影響………………………090
表4.8 農桿菌濃度與共培養天數對蝴蝶蘭癒合組織轉殖效率的影響…………091
表4.9 農桿菌濃度與共培養天數對蝴蝶蘭癒合組織轉殖效率影響的變方分析…092
表4.10 乙醯丁香酮濃度對蝴蝶蘭癒合組織應用農桿菌媒介法轉殖的影響…092
表4.11 蝴蝶蘭ATK032轉殖系於處理六個月後獲得潮黴素抗性癒合組織系的摘要……………………………………………………………………………093
表4.12 參試增幅核酸的引子序列…………………………………………………093
表4.13 蝴蝶蘭ATK019轉殖系再生植株進行聚合酶鏈鎖反應分析結果彙整094
表4.14 蝴蝶蘭ATL001轉殖系再生植株進行聚合酶鏈鎖反應分析結果彙整094
表4.15 蝴蝶蘭ATL003轉殖系再生植株進行聚合酶鏈鎖反應分析結果彙整094
表4.16 蝴蝶蘭ATL006轉殖系再生植株進行聚合酶鏈鎖反應分析結果彙整094
表4.17 蝴蝶蘭ATK016轉殖系再生植株進行聚合酶鏈鎖反應分析結果彙整095
表4.18 蝴蝶蘭ATK023轉殖系再生植株進行聚合酶鏈鎖反應分析結果彙整095
表4.19 蝴蝶蘭ATK032轉殖系再生植株進行聚合酶鏈鎖反應分析結果彙整096
表4.20 蝴蝶蘭ATK023轉殖系再生植株基因組DNA限制酶酶切後進行聚合酶鏈鎖反應分析結果彙整與比較……………………………………………096
表4.21 蝴蝶蘭ATK032轉殖系再生植株基因組DNA限制酶酶切後進行聚合酶鏈鎖反應分析結果彙整與比較……………………………………………097
表4.22 蝴蝶蘭ATK023轉殖系再生植株總量RNA進行反轉錄聚合酶鏈鎖反應分析結果彙整與比較…………………………………………………097
表4.23 蝴蝶蘭ATK032轉殖系再生植株總量RNA進行反轉錄聚合酶鏈鎖反應分析結果彙整與比較………………………………………………………098
表4.24 蝴蝶蘭ATK023轉殖系癒合組織進行聚合酶鏈鎖反應分析結果彙整與比較……………………………………………………………………………098
表4.25 蝴蝶蘭ATK032轉殖系癒合組織進行聚合酶鏈鎖反應分析結果彙整與比較……………………………………………………………………………099
表5.1 槍擊壓力與金粒子大小對蝴蝶蘭癒合組織表達GFP之影響……………128
表5.2 基因槍槍擊參數影響蝴蝶蘭癒合組織槍擊後1天表達GFP之變方分析…128
表6.1蝴蝶蘭轉殖植株葉圓片以不同GUS活性組織化學染色法處理1小時後，葉圓片呈色結果………………………………………………………………141
表6.2蝴蝶蘭轉殖植株葉圓片以不同GUS活性組織化學染色法處理6小時後，染色緩衝溶液的呈色結果……………………………………………………141
表7.1 蝴蝶蘭ATK32轉殖細胞系之GFP表達之相對值 …………………………161
表7.2 蝴蝶蘭ATK32擬轉殖株原生質體之GFP表達之相對值 …………………162
附表1 美國歷年蘭花類盆花銷售的數量與金額 ………………………………192
附表2 日本歷年蝴蝶蘭盆花銷售的數量與金額 …………………………………192
附表3 荷蘭歷年蝴蝶蘭盆花銷售的數量與金額 …………………………………193
附表4 臺灣歷年蝴蝶蘭產品進出口的總數量與總價值…………………………193
附表5 西元2006年臺灣蝴蝶蘭產品進出口數量與價值 ……………………193
附表6 西元2006年臺灣蝴蝶蘭活植株前十大出口國的出口價值與變動 …194
附表7 美國歷年蘭花植株前六大進口國的進口價值與變動 ……………194
附表8 蝴蝶蘭癒合組織誘導的培養基與培養條件……………………195
附表9 蝴蝶蘭癒合組織增殖的培養基與培養條件…………………..…196
附表10 蝴蝶蘭癒合組織再生的培養基與培養條件…………………………196
附表11中英名詞對照表……………………………….…………………197

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