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研究生:林督傑
研究生(外文):Tu-Chieh Lin
論文名稱:高雄二號茄懸浮細胞增殖及其體胚分化之研究
論文名稱(外文):Studies on Proliferation and Somatic Embryogenesis of Suspension Cells in Eggplant Solanum melongena cv. Kaohsiung 2
指導教授:林宜賢林宜賢引用關係鄭秋雄
指導教授(外文):Yi-Hsien LinChiu-Chsiung Cheng
口試委員:賴宏亮陳文輝
口試委員(外文):Horng-Liang LayWen-Huei Chen
口試日期:2017-07-28
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:植物醫學系所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2016
畢業學年度:105
語文別:中文
論文頁數:60
中文關鍵詞:茄(高雄二號茄)懸浮細胞細胞增殖細胞團分化體胚分化
外文關鍵詞:Solanum melongenacv."Kaohsiung2 "suspension cellsubculturecell proliferationcell cluster differentiationsomatic embryogenesis
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為利用高雄二號茄懸浮細胞於新抗病品系之篩選,懸浮細胞繼代培養及其體胚分化培養技術之建立十分關鍵。本研究探討供試植株齡期、培養基成分、供試植株栽培環境、癒傷組織繼代及癒傷組織來源對懸浮細胞增殖之影響;又,探討懸浮細胞來源、NAA濃度及植物生長調劑組成對懸浮細胞分化體胚之影響。比較60天、75天及90天株齡植株來源癒傷組織之懸浮細胞數,結果以60日齡者為最佳。懸浮細胞分別以MS、modified MS、CS-1培養基進行初代培養,結果各處理之細胞皆無法增殖。為增加建立培養獲得細胞數,將癒傷組織繼代後進行建立培養。結果以經繼代培養二次以上之癒傷組織進行建立培養可顯著增加懸浮細胞數。為尋找高增殖潛能之細胞來源,分別取不含葉脈葉塊、含支脈葉塊及主脈培植體誘導之癒傷組織作為細胞來源。。不同培植體來源之懸浮細胞增殖率以主脈最佳(310%)。進步調整癒傷組織及懸浮細胞具分化能力之條件,不同培植體來源之癒傷組織在含10 mg/l NAA之MS培養基中皆成功誘導體胚分化。懸浮細胞在混合使用NAA及kinetin條件下以含支脈之葉塊來源懸浮細胞可分化體胚。綜上所述,本研究最佳培養條件為:(1)在建立懸浮細胞上,以60日齡茄株主脈培植體來源並繼代二次以上之癒傷組織再於含10 mg/l NAA之MS培養基上可建立具有最佳增殖率之懸浮細胞。(2)在體胚分化上,以含支脈葉塊來源懸浮細胞於含8~14 mg/l NAA及0.1 mg/l kinetin之條件下可誘導體胚分化。
In this study, we attended to establish the techniques of preparing suspension cell from subculture and somatic embryogenesis of Solanum melongena cv. Kaohsiung 2. The effect on proliferation of the suspension cell, plant age, medium, cultivate environment of test plant, callus subculture and callus source were evaluated; effect on somatic embryogenesis of the suspension cell, the suspension cell source, NAA concentration and callus source were evaluated. In the comparison of the number of suspension cell suspended from the callus which induced from 60, 75, 90-day-old plants, 60-day-old plants produced 4.26106 cells/g callus, higher than other materials. Suspension cells were further cultured in MS, modified MS and CS-1 media to proceed primary culture, the results indicated that all of the treated suspension cells were not able to proliferate. In order to overcome the limit on proliferation, callus induced from cultivation rack as the explants were used and further compared the suspension cell number of subculture numbering. The callus had establish culture after subculture more than twice increased significantly in suspension cell number, which could get 1.16-1.35106 cells/g callus. For finding the suspension cells that have the good ability in proliferation, using the explants of leaf disc without vein or with vein and midrib as the suspension cell source. Compared proliferation rate of callus and suspension cell among each explants. Callus induced from the leaf disc with vein had the best result in callus proliferation (660%), but in suspension cell proliferation, the suspension cell induced from midrib was the best (310%). In order to get the callus and suspension cells that have good ability in somatic embryogenesis, callus, induced from the different explants, cultured on MS containing 10 mg/l NAA to differentiated somatic embryos. All treatments were successful in somatic embryogenesis. We tried to differentiate the somatic embryos from suspension cells with NAA alone or mixed with NAA and kinetin. The results indicated that there was not any somatic embryogenesis in NAA alone; in mixed NAA and kinetin, the suspension cells suspended from the leaf disc with vein in different concentrations could differentiate the somatic embryos. In this study, it’s found that (1) In suspension cell proliferation, the suspension cells suspended from callus which induced from the midrib of 60-day-old plants offered as the explants and subculture more than twice, cultured in MS liquid medium containing 10 mg/l NAA had the best result in proliferation rate. (2) In somatic embryogenesis of suspension cells, the suspension cells suspended from the leaf disc with vein cultured in MS medium containing 8-14 mg/l NAA and 0.1 mg/l kinetin could successfully differentiated somatic embryos.
摘要 I
Abstract III
目錄 V
圖表目錄 IX
壹、 前言 1
貳、 前人研究 3
一、 茄子癒傷組織誘導 3
二、 茄子懸浮細胞培養 5
三、 茄子植株再生 7
參、 材料與方法 11
一、 供試植株之培育 11
二、 供試材料消毒及培植體來源 11
三、 培養基製備 11
四、 癒傷組織誘導及繼代培養 12
五、 懸浮細胞建立培養、初代培養及繼代培養 12
六、 體胚分化培養 13
七、 數據回收及分析 13
八、 供試植株株齡對茄子懸浮細胞建立培養之影響 14
九、 培養基對茄子懸浮細胞初代培養細胞增殖率之影響 14
十、 供試植株栽培環境及癒傷組織繼代培養對懸浮細胞建立培養懸浮細胞數之影響 14
十一、 培植體對誘導癒傷組織及其增殖之影響 14
十二、 癒傷組織來源及癒傷組織鮮重對懸浮細胞建立培養懸浮細胞數之影響 15
十三、 細胞來源對懸浮細胞增殖率之影響 15
十四、 癒傷組織來源對癒傷組織分化體胚之影響 15
十五、 細胞來源及NAA濃度對茄子懸浮細胞分化細胞團之影響 15
十六、 植物生長調節劑組成及培養方式對不同來源懸浮細胞分化細胞團之影響 16
肆、 結果 17
一、 供試植株株齡對茄子懸浮細胞建立培養之影響 17
二、 培養基對茄子懸浮細胞初代培養細胞增殖率之影響 17
三、 供試植株栽培環境及癒傷組織繼代對茄子細胞懸浮培養之影響 17
四、 培植體對誘導癒傷組織及其增殖之影響 17
五、 癒傷組織來源及癒傷組織鮮重對茄子懸浮細胞建立培養懸浮細胞數之影響 18
六、 細胞來源對茄子懸浮細胞增殖率之影響 18
七、 癒傷組織來源對茄子癒傷組織分化體胚之影響 19
八、 細胞來源及NAA濃度對茄子懸浮細胞分化細胞團之影響 19
九、 植物生長調節劑組成及培養方式對不同來源懸浮細胞分化細胞團之影響 19
伍、 討論 21
陸、 結論 26
柒、 參考文獻 28
捌、 圖表 35
玖、 附錄 60
壹拾、 謝誌 63
壹拾壹、 作者簡介 64
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