臺灣博碩士論文加值系統

紅豆是世界上第12大豆科作物，雖然並非主要的糧食作物，但在亞洲紅豆是被廣泛應用在食品加工的作物。早先，利用切離的上胚軸進行農桿菌基因轉殖研究，GUS基因的表現無礙；但是，胚軸切面細胞黃化及壞死情形嚴重，使得農桿菌基因轉殖效率降低。本研究利用紅豆高雄6號(KS6)、高雄7號(KS7)、高雄8號(KS8)及高雄9號(KS9)等四個品種，於黑暗環境發芽生長七天的上胚軸作為培植體。使用帶有pCAMBIA1201或1303質體的不同菌系農桿菌，包括分別為EHA105、LBA4404及KYRT1進行轉殖研究，嘗試找出適合紅豆胚軸轉殖的相關條件。
適合紅豆胚軸轉殖的相關條件研究結果顯示：(1)帶有pCAMBIA1201的農桿菌感染效果較pCAMBIA1303效果來得好。(2)在紅豆基因轉殖的系統中，EHA105菌系的感染效果較KYRT1及LBA4404來得高。(3)以EHA105-pCAMBIA1201來感染紅豆培植體，GUS基因表現以高雄9號為最佳，感染面積可達67.62%，但黃化情形為69.71%較為嚴重；高雄6號與高雄8號的感染強度較廣，分別為70.31%與73.22，高雄6號木質素的累積情況低，而高雄8號的切面細胞黃化的程度較低。

Azuki bean (Vigna angularis (Willd.) Ohwi and Ohashi) is one of the twelve most important grain legumes in the world. In East Asia, the azuki bean is almost applied to food processing extensively. Agrobacterium- mediated gene transfer using azuki bean hypocotyls showed normal transient GUS gene expression, Agrobacterium-induced hypersensitive necrotic reaction in plant cells are seriously problems. Four cultivars of Azuki bean, Kaohsiung NO.6 (KS6), KS7, KS8 and KS9 were tested for plant regeneration by using explants sections from the epicotyls formed the seeds germinating seven days after planting in the dark. It is the Agrobacterium to use different strain with plasmid pCAMBIA 1201 or 1303, including transformation research for EHA105, LBA4404 and KYRT1, try to find out the relevant condition that the suitable azuki bean epicotyls transfer to transformation.
The result of the study shows that the Agrobacterium carrying pCAMBIA1201 had higher efficiency than pCAMBIA1303. In gene transfer system of azuki bean, the infection efficiency of EHA105 strain is higher than KYRT1 and LBA4404. Using EHA105- pCAMBIA1201 to infect the explants of azuki bean shows that the KS9 had the biggest GUS gene expression extent, which is up to 67.62%, however, the condition of brownish is the most serious of all. Moreover, the intensity of GUS gene expression of KS6 and KS8 are the strongest, 70.31% and 73.22, respectively. In addition, the accumulation of lignin in KS6 is lower than the other one; the brownish of section cell in KS8 is lower, too.

中文摘要 ……………………………………………………………… Ⅰ
英文摘要 ……………………………………………………………… Ⅱ
本文目次 ……………………………………………………………… Ⅳ
圖 目 錄 ……………………………………………………………… Ⅵ
表 目 錄 ……………………………………………………………… Ⅸ
縮 寫 表 ……………………………………………………………… Ⅹ
第 一 章 緒論 ………………………………………………………… 1
1-1 紅豆簡介 ……………………………………………… 1
1-2 紅豆的營養成分與應用 ……………………………… 2
1-3 紅豆在台灣的發展 …………………………………… 2
1-4 植物基因轉殖系統 …………………………………… 3
1-5 影響農桿菌轉殖之因子 ……………………………… 8
1-6 豆科植物基因轉殖 …………………………………… 12
1-7 研究動機 ……………………………………………… 14
第 二 章 材料與方法 ………………………………………………… 15
2-1 勝任細胞(competent cell)的製備……………………… 15
2-2 農桿菌的轉型實驗(transformation)…………………… 16
2-3 質體DNA鑑定………………………………………… 17
2-4 鏈合酶聚合反應偵測 ………………………………… 18
2-5 農桿菌生長曲線 ……………………………………… 20
2-6 植物實驗材料處理 …………………………………… 21
2-7 農桿菌轉殖 …………………………………………… 22
2-8 木質素偵測 …………………………………………… 23
2-9 GUS基因表現 ………………………………………… 24
2-10 影像處理 …………………………………………… 24
第 三 章 實驗結果 …………………………………………………… 26
第 四 章 討論 ………………………………………………………… 33
第 五 章 結論 ………………………………………………………… 37
參考文獻 ……………………………………………………………… 38

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