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研究生:簡瑋廷
研究生(外文):Wei Ting Jian
論文名稱:新鐵砲百合(Lilium×formolongihort.)之體胚發生與大量繁殖
論文名稱(外文):SOMATIC EMBRYOGENESIS AND MASS PROPAGATION OF LILIUM ×FORMOLONGI HORT.
指導教授:何錦玟
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:72
中文關鍵詞:新鐵砲百合體胚發生大量繁殖
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中文摘要
本研究利用液體培養中的體胚發生,進行新鐵砲百合(Lilium formolongi hort.)大量繁殖。新鐵砲百合”諾利庫拉”品種由試管內實生苗鱗片,扦插於含0.1 mg/l NAA、90 g/l Sucorse的MS培養基,誘導癒合組織。癒合組織細胞液體培養的最適條件為,細胞接種量為3.0 g/20 ml,培養於含0.1 mg/l NAAmg/l與Sucorse 30 g/l的MS培養基,每培養四週細胞鮮重可以倍增。液體培養可得到大量小細胞團,經透明法與埋蠟切片觀察,有完整的表皮與封閉的微管束,具有體胚的結構。液體培養之細胞移至0.1 mg/l Kinetin、0.1 mg/l NAA與Sucorse 30 g/l的MS培養基,有最高的胚芽轉換率76.67 %。1 g的癒合組織細胞在培養一年後可以得到1.16×105個植株。再生的植株染色體倍數2n=24,與母本相同,定植後能正常開花。液體培養之細胞於氣舉攪拌與Super Spinner生物反應器培養,均因剪應力而造成細胞褐變死亡。
由種子直接誘導五個新鐵砲百合與1個台灣百合(L. formosanum wall.)的胚性癒合組織,6個百合品種中有4品種得到共9個具有良好再生能力的細胞系,可以在液體培養中不但得到分離的細胞團塊,且植株再生能力良好,亦可利用於液體培養大量增殖。

ABSTRACT
We investigated the somatic embryogenesis on friable callus of Lilium×formolongi cv.”Norikula” in suspension culture. The in vitro bulb was developed from sowed seeds on MS basal medium supplement with 30 g/l sucrose. The meristematic callus of L.×formolongi cv.”Norikula” were proliferated from in vitro bulb scales placed on MS basal medium supplement with 0.1 mg/l NAA, 90 g/l sucrose and 0.8 % Agar. Cell suspension cultures were established for embryogenesis in MS basal medium supplemented with 0.1 mg/l NAA and 90 g/l sucrose. We obtained a lot of somatic embryos from suspension culture of meristematic callus. The structure of somatic embryo was proved by tissue anatomy.
The optimal medium for suspension culture was MS basal medium supplemented with 0.1 mg/l NAA and 30 g/l sucrose, and the cell fresh weight could be double every 4 weeks. Maximum somatic embryos regeneration ratio was achieved 76.67 % in MS basal medium supplemented with 0.1 mg/l NAA, 0.1 mg/l kinetin and 30 g/l sucrose. A total of 1.16 ×105 plants can be produces from 1 g (fresh weight) callus through somatic embryogenesis. Plantlets derived from somatic embryo were diploid (2n=24) and flowering after transfer to soil for 9 months. Cell clumps cultured in Super Spinner bioreactor and air lift stir bioreactor for mass propagation were turned brown and dead by shear stress.
6 cell-lines from 3 species of L.×formolongi were collected and 3 cell-lines from L. formosanum were collected, too. All of those cell-lines were grown well and obtained meristematic nodular callus from suspension culture.

目 錄
ACKNOWLEDGMENTS iii
中文摘要 iv
ABSTRACT v
LIST OF TABLES x
LIST OF FIGURES xiii
ABBREVIATION xix
一、前人研究 1
1.1 新鐵炮百合簡介 1
1.2 植物組織培養在百合上的研究 1
1.2.1 群間雜交品種的育種 2
1.2.2 種苗大量繁殖 2
1.2.2.1 生長點培養 2
1.2.2.2 不定芽增殖 3
1.2.2.3 癒合組織培養 3
1.3 新鐵砲百合組織培養的相關研究 5
二、材料與方法 7
2.1 材料來源 7
2.2 培養基 7
2.3 培養環境 8
2.4 由無菌小苗誘導癒合組織 8
2.5液體培養與建立 9
2.6 體胚之再生及體胚轉化率之計算 9
2.7 體胚型態觀察 10
2.7.1 透明法 10
2.7.2 埋蠟切片法 10
2.8 染色體倍數檢查 11
2.9 出瓶馴化 11
2.10 細胞生長曲線測試 11
2.11 最適接種量測試與蔗糖濃度對細胞鮮重增加的影響 12
2.12 BA、kinetin與糖濃度對液體培養細胞鮮重增加的影響 12
2.13 生物反應器培養 12
2.13.1 Super Spinner bioreacter 12
2.13.2 氣舉攪拌式生物反應器 12
2.14 統計分析 13
三、結果 21
3.1由無菌小苗誘導癒合組織 21
3.2 種子直接誘導癒合組織 26
3.2.1 ”諾利庫拉”品種 26
3.2.2 ”雷山一號” 30
3.2.3 ”雷山三號” 34
3.2.4 ”雷山極早生” 34
3.2.5 ”奧萬大” 37
3.2.6 ”白蘭莎” 37
3.2.7 再生良好之細胞系液體培養 40
3.3 體胚型態觀察 46
3.4 體胚再生植株染色體檢查 46
3.5 馴化與定植 46
3.6 細胞生長曲線 54
3.7 液體培養的最適接種量與最適蔗糖濃度 57
3.8 BA、kinetin與糖濃度對細胞的影響 60
3.8.1 BA與Kinetin的添加對細胞鮮重的影響 60
3.8.2 BA與Kinetin的添加對似胚體比率的影響 60
3.8.3 BA與Kinetin的添加對胚芽轉換率的影響 66
3.9.4體胚的大量增殖 68
四、討論 69
4.1 癒合組織的誘導 69
4.2 液體培養中的生長曲線 70
4.3 液體培養的最適接種量與最適蔗糖濃度 71
4.4 BA、kinetin與糖濃度對細胞的影響 71
4.5 生物反應器培養 72
4.6 體胚型態觀察與染色體檢查 73
五、結論 74
六、參考文獻 75

六、參考文獻
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