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研究生:郭俊緯
研究生(外文):Chun-Wei Kuo
論文名稱:香蕉體胚衍生胚性細胞懸浮培養、胚性細胞極性誘導與體胚發生
論文名稱(外文):The Somatic Embryo-Derived Cell Suspension Culture, Polar Induction and Somatic Embryogenesis of Triploid Bananas
指導教授:許圳塗許圳塗引用關係
指導教授(外文):Chou Tou Shii
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:園藝學研究所
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:89
中文關鍵詞:胞外酸鹼值極性硝酸態氮銨態氮
外文關鍵詞:extracellular pHpolarityNO3-NNh4-N
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香蕉Musa AAB cv. Raja成熟體胚藉由液體震盪懸浮培養,釋放胚性細胞,建立胚性細胞懸浮培養。參試培養基不同氮源比例及不同濃度2,4-D處理。在全量硝酸態氮與單一銨態氮下無法獲得胚性細胞。兩氮源組合含1 mg/L 2,4-D之MS培養基,培養9週後可得到均質胚性懸浮細胞系。高2,4-D(4 mg/L)有誘導胚性細胞發育為球狀體,低2,4-D(1 mg/L)維持胚性細胞非極化增生。體胚培殖體建立懸浮細胞比以幼花序培殖體,可縮短2到4個月的時間。體胚衍生胚性懸浮細胞長期繼代培養,氮型比會影響胞外pH值及細胞發展形態。硝酸與銨氮比2:1處理,胞外pH自調為5.0-5.6,同時易極化生長大量球胚。1:1與1:2,胞外pH分別在4.4-4.6及3.8-4.2,形成大量胚性細胞團。顯示高銨態氮比例,有利降低胞外pH值,同時促使維持胚性細胞非極化增生,形成前胚性細胞團。
Musa AAA cv. Robusta胚性細胞酸化處理(pH 3.5)促進前胚性細胞團解離,釋放游離胚性細胞,調控pH 4.8處理則誘導胚性細胞朝向前胚發生。Musa AAB cv. Raja與 Musa AAA cv. Robusta 的胚性細胞系調控pH高於4.8,可同步誘導胚性細胞不對稱分裂,其雙胞大小比值為1.2。14天時Raja則可發育具有原表皮之球狀體胚,而Robusta則不易分化具有原表皮之原胚。相對地,在控制低pH值下,則其前胚性定向細胞呈對稱分裂為主,雙胞大小比值為1.04。
Musa AAA cv. Robusta酸化前處理4、7、14天的胚性細胞系,再處理pH 4.8。酸化前處理過久,體胚發生時胚性細胞發育球胚會受到干擾。酸化前處理4天發育具有原表皮的完整球胚,但是酸化前處理7、14天的球胚外觀不完整。球胚經平板培養分化體胚,酸化前處理7、14天的球胚大量褐化,無法產生體胚。酸化前處理4天有大量的體胚發生,與對照組比較。對照組由胚性細胞團發生的體胚,叢生狀生長,體胚發育緩慢且大小不一,多數直徑小於0.5 mm。經酸化前處理4天同步誘導球胚發育,體胚個別化生長,生長較一致,直徑多大於1 mm。藉由pH位階進行酸化解離細胞團,同步高pH誘導球胚發育,Musa AAA cv. Robusta可生長個別化與發育較一致 的體胚。
Embryogenic cell suspensions of Musa AAB cv. Raja were obtained by releasing embryogenic cells from somatic embryos in liquid MS medium with various nitrate/ammonium ratios, 4 mg/L 2, 4-D and 1 mg/L 2, 4-D. Embryogenic cells could not obtain successfully in MS media with full nitrate and ammonium. Nitrate and ammonium must supply simultaneously despite the nitrate/ ammonium ratios and all obtained embryogenic cells in 9 weeks faster about 2 to 4 months than embryogenic cell suspensions derived from male flowers. Somatic embryogenesis was induced at high 2, 4-D but embryogenic cells dedifferentiating and proliferating at low 2, 4-D. Embryogenic cell suspensions derived from somatic embryos subculture in MS media. Regulation of extracellular pH in cell suspensions was successful by modifying nitrate/ammonium ratio. Cell suspensions in media with nitrate/ammonium ratio 2: 1 were about pH 5.0-5.6 and had large quantities of globoids. Cell suspensions of nitrate/ammonium ratios 1: 1, 1: 2 were about 4.4-4.6 and 3.8-4.2, which were proliferous. High ammonium concentration could lower extracellular pH and promote non-polar growth to form embryogenic cell masses.
Acidic treatment (pH 3.5) promoted preembryogenic masses to disunite small masses and release single cells. After acidic treatment for 4, 7 and 14 days, Cell suspensions could develop globoids at pH 4.8. Embryogenic cells proceeded to do asymmetric division at or over pH 4.8. The cell size ratio of bicell of Musa AAA cv. Robusta and Musa AAB cv. Raja both was at or over 1.2. After 14 days, the globoids of Musa AAB cv. Raja developed completely. The proembryos of Musa AAA cv. Robusta could not develop protoderm successfully. Oppositely, the extracellular pH was at or fewer than 4.6 and division of embryogenic cells was symmetric. The cell size ratio of bicells was about 1.04.
Cell suspensions of Musa AAA cv. Robusta titrated to pH 4.8 after acidic treatment for 4, 7 and 14 days. Only acidic treatment for 4 days could differentiate protoderm successfully and develop globoids, proembryos of acidic treatment for 7 and 14 days were failed. Plating culture of acidic treatment for 7 and 14 days turned brown and failed to differentiate somatic embryos. Plating culture of CK and acidic treatment for 4 days produced large quantities of somatic embryos. Somatic embryos derived from preembryogenic masses of CK grew in turf, developed asynchronously and mostly smaller than 0.5 mm. Somatic embryos derived from globoids of acidic treatment for 4 days were individual, growth uniformly and mostly larger than 1 mm.
壹、 前言(Introduction)……………………………..………1
貳、 前人研究(Review)
一、 體胚發生………………………………………………..3
二、 懸浮培養………………………………………………..7
三、 胚性細胞發育相…………….…………………………9
四、 胞外酸鹼值與細胞培養……………………………..10
五、 極性誘導………………………………………………13
參、 材料與方法(Materials and Methods)
(一) 胚性懸浮細胞之建立………………………………...16
(二) 胚性懸浮細胞系維持培養與生長………………….18
(三) 胚性細胞極化誘導與前胚發生……………………..19
肆、 結果(Results)
(一) 胚性懸浮細胞之建立………………………………...22
(二) 胚性懸浮細胞系維持培養與生長相分析………….24
(三) 胚性細胞極化誘導與前胚發生……………………..26
伍、 討論(Discussion)
(一)探討體胚衍生胚性細胞懸浮培養之影響因素..……...69
(二)香蕉胚性細胞高pH誘導極性建立.………………….73
(三)Musa AAA cv. Robusta pH位階調控與體胚發生…..75
陸、 中文摘要…………………………………...…………..77
柒、 Summary…………………………………………………..79
捌、 參考文獻(References)…………………………………81
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