臺灣博碩士論文加值系統

中南美洲原生觀賞用鳳梨與臺灣重要商業品種之遺傳親緣性分析
本試驗以市場上商業價值較高的7屬18種觀賞鳳梨為材料，進行逢機增殖DNA片段(Randomly Amplified Polymorphic DNA, RAPD)的分析。由20個RAPD 引子所產生的條帶之中，共獲得119個具多型性的分子標誌。在屬間鑑別上，4個為Cryptanthus屬所特有，3個為Dyckia屬所特有，2個為Guzmania屬所特有，12個為Neoregelia屬所特有，1個為Tillandsia屬所特有，1個為Vriesea屬所特有。在種間鑑別上，30個多型性的分子標誌為各種間所特有之差異條帶，其它則為多型性條帶，可以用來分辨不同的物種，尤其是外觀相似的不同物種。利用Jaccard方法所求出不同樣本之間的相似度及UPGMA方法所建立出來的親緣關係圖，除結果顯示與形態分類學一樣之外，本試驗結果所得之屬與種專一性分子標誌更可提供日後觀賞鳳梨栽培品種鑑別良好工具。
擎天屬龍鳳品種之細胞培養與體胚再生
以觀賞鳳梨擎天屬龍鳳品種未成熟之小花附屬器官作為初級培殖體，接種於半固體培養基1/2MS添加Citric acid 0.1 mg/ml 、2,4-D 0.2-1.5 mg/L或合併含有NAA 0.5 mg/L，以幼花瓣培殖體癒合組織發生率可達80%，逆分化效果佳。2-3個月逆分化為癒合組織並出現有原胚狀體。將胚性癒合組織移置於TB5添加2,4-D 0.5 mg/L的液態增生培養基中，以110 rpm懸浮振盪培養，約4個月可得到均質之胚性細胞系。懸浮細胞族群經篩選後，平板培養於培養皿，以SH半固體培養基參試生長素及細胞分裂素組合，其中組合低量0.2 mg/L NAA + 0.2 mg/L 2ip +0.1 mg/L kinetin可誘導較多之胚狀體，並成熟抽出芽體。由1/30 ml PCV胚性細胞團，平均可再生200-400芽體，若將生長素省略培養生成之胚苗較易有畸形芽體。將成熟胚狀體或芽體移至1/2SH補充0.2 mg/L NAA + 0.2 mg/L 2ip固態培養基中，則可長出根系良好之小植株，移出瓶外健化處理，成活率可達近100%。

Genetic Relationship between Ornamental Bromeliads of Centric South America and Commerical Cultivars in Taiwan.
The RAPD polymorphic markers were used to analyze the genetic similarity among eighteen collected accessions and cultivars attributed to seven genera of Bromeliaceae. A total of 119 markers were obtained from 20 RAPD primers. In genus identification, there are four, three, two, twelve, one, and one markers specific to Cryptanthus, Dyckia, Guzmznia, Neoregelia, Tillandsia, and Vriesea, respectively. Besides, all the eighteen tested accessions could be identified with thirty proposed markers. The result suggests that these thirty markers are useful tools for genera and species or cultivars discrimination, especially for species with high morphological similarity. The dendrogram established by Jaccard and UPGMA methods is coincident with the taxonomy. Moreover, our studies suggest a potential molecular marker to identify the cultivars of Bromeliaceae.
Somatic Embryogenesis in Cell Suspension Culture of Ornamental Bromeliads.
The various parts in young floret of ornamental bromeliad Guzmania cv. Cheery were used as explants inoculated on 1/2 MS medium supplemented with citric acid 0.1 mg/ml and 2,4-D 0.2-1.5 mg/L combinated with NAA 0.5 mg/L. The petal explants were potential to dedifferentiate embryogenic callus as high as 80 % by 2-3 months after culture. The callus with proembryoids were transferred into TB5 liquid medium added with 2,4-D 0.5 mg/L, cultured on 110 rpm shaker. The heterogeneous suspension cells were achieved about 2 months after suspension culture, which were competent to proliferate in apolar growth cycle, and formation of amorphic preembryogenic masses. The screened PEMs were plated on filter paper or gellified stratum enriched with half storgth basal salt of SH medium supplemented with NAA 0.2 mg/L, 2ip 0.2 mg/L and kinetin 0.1 mg/L, and induced multiple somatic embryo formation in each mini-callus. The mature somatic embryos were initially to in sprout, and acquired 200-400 shoots per dish inoculated with 1/30 PCV. The somatic embryos or emerging shoots were transferred on 1/2 SH medium after with NAA 0.2 mg/L and 2ip 0.2 mg/L benefit to converte into vigorous plantlets. The survival in exvitro transplant of the plantlets approached close to 100 % under mist propagation condition.

內 容 目 次
一、 前言..………………………………………………………………………...1
二、 前人研究..…………………………………………………………………...3
(一) 鳳梨科植物之遺傳親緣性分析…………………………………………3
(二) 鳳梨科植物之體外繁殖…………………………………………………6
A. 無菌播種……………………………………………………………...6
B. 短縮莖培養…………………………………………………………...7
C. 葉片培養……………………………………………………………...8
D. 組培苗根培養………………………………………………………...9
E. 花器培養…………………………………………………….………..9
三、 材料與方法………………………………………………………………...12
(一) 中南美洲原生觀賞用鳳梨與臺灣重要商業品種之遺傳親緣性分析..12
A. 參試材料…………………………………………………………….12
B. 基因組DNA的抽取………………………………………………...12
C. 引子篩選………………………………………….…………………13
D. PCR (Polymerase Chain Reaction) 聚合反應………………………13
E. 資料分析…………………………………………………….………14
(二) 擎天屬龍鳳品種之細胞培養與體胚再生……………………………..15
A. 參試材料…………………………………………………………….15
B. 取材與消毒……………………………………………….…………15
C. 試驗內容………………………………………….…………………15
(1) 花器培養逆分化胚性癒合組織之誘導………………………..15
(2) 胚性細胞懸浮再生系統之建立………………………………..16
(3) 平板培養………………………………………………………..16
(4) 小植株建立……………………………………………………..16
(5) 移植……………………………………………………………..17
四、 結果………………………………………………………………………...25
(一) 中南美洲原生觀賞用鳳梨與臺灣重要商業品種之遺傳親緣性分析..25
(二) 擎天屬龍鳳品種之細胞培養與體胚再生……………………………..28
(1) 花器培養逆分化胚性癒合組織之誘導………………………..28
(2) 胚性細胞懸浮再生系統之建立………………………………..29
(3) 平板培養………………………………………………………..30
(4) 小植株建立……………………………………………………..31
(5) 移植……………………………………………………………..32
五、 討論………………………………………………………………………...54
(一) 中南美洲原生觀賞用鳳梨與臺灣重要商業品種之遺傳親緣性分析..54
(二) 擎天屬龍鳳品種之細胞培養與體胚再生……………………………..57
(1) 花器培養逆分化胚性癒合組織誘導之影響因子……………..57
(2) 胚性細胞懸浮再生系統之建立………………………………..58
(3) 平板培養………………………………………………………..61
(4) 胚苗轉換………………………………………………………..62
(5) 移植……………………………………………………………..63
六、 中文摘要…………………………………………………………………...64
(一) 中南美洲原生觀賞用鳳梨與臺灣重要商業品種之遺傳親緣性分析..64
(二) 擎天屬龍鳳品種之細胞培養與體胚再生……………………………..65
七、 英文摘要…………………………………………………………………...66
(一) 中南美洲原生觀賞用鳳梨與臺灣重要商業品種之遺傳親緣性分析..66
(二) 擎天屬龍鳳品種之細胞培養與體胚再生……………………………..67
八、 參考文獻…………………………………………………………………...68
九、 附表(龍鳳擎天鳳梨癒合組織、細胞懸浮培養及體胚發生之基本培養基
組成)………………………………………………………………………73
圖 目 錄
圖 1. RAPD分析參試之18種觀賞鳳梨樣品植株及其開花形態……………18
圖 2. 觀賞鳳梨龍鳳擎天開花特性(A)及其小花花器構造(B)………………...24
圖 3. 十八種觀賞鳳梨以OPB-5引子進行RAPD分析所得之條帶型………..33
圖 4. 十八種觀賞鳳梨樣本RAPD分析所得到的叢群分析樹狀圖…………...34
圖 5. 利用RAPD分子標誌相似性分群所得之觀賞鳳梨在中南美洲地理分佈圖…………………………………………………………………………..35
圖 6. 龍鳳擎天鳳梨花器培養誘導癒合組織生長情形………………………..39
圖 7. 龍鳳擎天鳳梨花瓣衍生之胚性癒合組織在 TB5 液體培養基細胞懸浮培養生長情形……………………………………………………………..41
圖 8. 觀賞鳳梨球體期之胚性懸浮細胞系以平板培養誘導體胚發生之情形..43
圖 9. 龍鳳擎天鳳梨平板培養叢生胚萌芽情形………………………………..45
圖 10.體胚苗健化培養及溫室生長情形………………………………………..46
圖 11.觀賞鳳梨胚性細胞再生系統……………………………………………..48
圖 12.外控胞外pH對細胞生長之影響…………………………………………49
表 目 錄
表 1. 參試18種觀賞鳳梨樣品及其原生地…………………………………….23
表 2. 適合觀賞鳳梨RAPD分析之20個引子序列……………………………..36
表 3. 參試觀賞鳳梨RAPD共同標誌、屬間及種間專一性分子標誌………….37
表 4. 十八種觀賞鳳梨物種之RAPD條帶以Jaccard方法分析所得之相似度..38
表 5. 2,4-D與NAA對觀賞鳳梨“Cherry”花器培養誘導癒合組織之效應….51
表 6. 2,4-D與椰子水對觀賞鳳梨“Cherry”懸浮細胞培養之影響……………...52
表 7. NAA、2ip及kinetin對觀賞鳳梨“Cherry”平板培養胚體生長之影響……55

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