臺灣博碩士論文加值系統

摘 要
台灣雲杉成熟胚可以成功的誘導出胚原性組織（embryogenic cultures），其中二個不同的細胞株（cell line T2及cell line T4）在體胚成熟分化時的能力不同，在ABA（abscisic acid）處理之後T2細胞株具體胚成熟能力及體胚產量高，而T4細胞株則甚少有體胚成熟之反應。在經HPLC（high performance liquid chromatography）檢測後，T4細胞在增殖期的IAA（indole-3-acetic acid）含量比T2細胞高出7.5倍，經ABA處理後T4細胞的IAA含量卻比T2細胞低。二個細胞株在增殖期經抗生長素（antiauxin）TIBA（2,3,5-triiodobenzoic acid）或PCIB（2-(4-chlorophenoxy)-2-methylpropionie acid）處理不同濃度及時間，以TIBA 1 µM或PCIB 5 µM，處理時間8週，可改善T4細胞株的體胚成熟分化能力，明顯提高T4細胞株stage 2的體胚產量。T4細胞株經此抗生長素處理後，亦驗證得知其IAA的含量有明顯的下降，證明IAA的含量與體胚的成熟分化有相關性。
關鍵詞：胚性-懸柄細胞團、吲哚乙酸、生長素、抗生長素、高效能液相層析儀

Abstract
Embryogenic cell culture lines T4 and T2（control）have been initiated from two mature zygotic embryos of Picea morrisonicola Hay.. Cell line T2 was capable of producing mature somatic embryos under ABA treatment. Cell line T4 was difficult to differentiate into mature somatic embryos under the same ABA treatment. The HPLC analysis of endogenous IAA content in these two culture lines at the proliferation stage confirmed that the cell line T4 produced IAA at about 7.5 times greater than that of line T2. Cell line T4, however, produced much lower IAA than those in line T2 after ABA incubation. An antiauxin, 2,3,5-triiodobenzoic acid（TIBA）or 2-(4-chlorophenoxy)-2-methylpropionie acid（PCIB）can improve cell line T4 to develop mature somatic embryos. Either 1 µM TIBA or 5 µM PCIB can increase the production of stage 2 embryo in T4 cell line when culture was treated in proliferation stage for 8 weeks. Both treatments can decrease IAA levels, significantly. These results indicate that the IAA contents will highly manipulate the process of somatic embryo maturation.
Key words：embryonal-suspensor mass、IAA、auxin、antiauxin、HPLC、high performance liquid chromatography

目 錄
頁數
一、前言… … … … … … … … … … … … … … … … … … … … 1
二、前人研究… … … … … … … … … … … … … … … … … … …5
三、材料與方法… … … … … … … … … … … … … … … … … … 9
（一）材料… … … … … … … … … … … … … … … … … …9
（二）不同培養階段細胞內生生生長素之分析… … … … …11
1.內生生長素的測定… … … … … … … … … … …11
2.IAA萃取物的純度追蹤… … … … … … … … … … … 13
（三）抗生長素對體胚成熟之影響… … … … … … … … …15
1.增殖期的胚原性組織染色 … … … … … … … …16
2.成熟期的細胞團體胚分化… … … … … … … … … … 17
（四）抗生長素處理後之內生生長素的分析 … … … … … …19
（五）試驗設計與資料分析… … … … … … … … … … … 19
（六）培養條件… … …… … … … … … … … … … … … 20
四、結果… … … … … … … … … … … … … … … … … … … …21
（一） 細胞團外觀及染色觀察… … … … … … … … … …21
1.細胞團外觀… … … … … … … … … … … … …21
2.染色觀察… … … … … … … … … … … … … 23
（二）不同培養階段細胞內生生長素分析之結果… … … … … 25
1.內生生長素的測定… … … … … … … … … … … …25
2.IAA萃取物的純度追蹤… … … … … … … … … … 28
（三）抗生長素對體胚成熟之影響… … … … … … … … …32
1.增殖期的胚原性組織染色觀察 … … … … … …32
2.成熟期Ⅰ的細胞團體胚分化能力… … … … … … … …34
3.成熟期Ⅱ的細胞團體胚分化能力 … … … … … … …36
（四）抗生長素處理後之內生生長素的分析 … … … … … …41
五、討論… … … … … … … … … …… … … … … … … …43
六、參考文獻… … … … … … … … … …… … … … … … …51
七、附錄… … … … … … … …… … … … … … … … 61

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