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研究生:蔡顓印
研究生(外文):Chuan-Yin Tsai
論文名稱:水稻台稉9號癒傷組織及細胞培養之研發及其應用
論文名稱(外文):Development and Application of Callus and Cell Culture of Oryza sativa var. japonica Taiken 9
指導教授:鄭秋雄
指導教授(外文):Chiu-Chsiung, Cheng
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:熱帶農業暨國際合作系所
學門:農業科學學門
學類:一般農業學類
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:90
中文關鍵詞:水稻台稉9號組織培養癒傷組織細胞培養植株再生白葉枯病菌白葉枯病抗病篩選抗白葉枯病細胞系
外文關鍵詞:Oryza sativa var. japonica Taken 9tissue culturecalluscell cultureplantlet regenerationbacterial leaf blightXanthomonas oryzae pv. oryzaedisease-resistant selectionbacterial leaf blight-resistant cell lines
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本研究之目的首為研發水稻癒傷組織誘導培養、細胞培養及植株再生之培養系統,進一步應用於進行抗白葉枯病細胞系之篩選。以水稻台稉9號成熟種子為培植體,探討培養基、水晶洋菜含量、蔗糖含量及植物生長調節劑對誘導水稻台稉9號種子癒傷組織之影響。結果以含2 mg/l 2,4-D、3%蔗糖、0.4%水晶洋菜之CS-1培養基置於500 lux光照下培養45天之癒傷組織形成量最佳,單位培植體種子之癒傷組織鮮重約0.334 g。另,觀察2,4-D含量(0.5-5.0 mg/l)對癒傷組織顆粒大小與質地之影響,則顆粒大小會隨2,4-D含量之提高而逐漸變小,但球形顆粒狀、球形團粒與整體狀等質地在所有處理中均有發生。細胞培養則以含2 mg/l之2,4-D之CS-1培養基誘導45天之癒傷組織為材料,並懸浮細胞建立培養第10天之懸浮細胞數最多,每3 g鮮重癒傷組織可獲得約1.4×106個細胞。探討培植體癒傷組織鮮重、植物生長調節劑、光照度、水晶洋菜含量對癒傷組織植株再生之影響,結果以含2 mg/l之 NAA及4 mg/l之kinetin、3%蔗糖、0.6%水晶洋菜之CS-1培養基,並置於3500 lux光照下之植株再生率最佳,約60%。以上述培養細胞誘導條件所誘導之懸浮細胞為供試材料,以白葉枯病菌為篩選劑,利用懸浮細胞與白葉枯病菌共同培養法進行抗白葉枯病細胞系之篩選,將篩選之抗白葉枯病細胞系與水稻台稉9號培養細胞分別處理白葉枯病菌培養過濾液,結果其細胞殘存率分別為96%與50.4%,顯示篩選之抗白葉枯病細胞系對病菌培養過濾液具有抗性。抗白葉枯病細胞系連續繼代培養於含2 mg/l 2,4-D之CS-1培養基中可分化出細胞團,細胞團亦可分化出體胚,但體
胚無法持續成長為植株。
The purposes of this study are first to develop the culture systems of callus induction, cell culture and plantlet regeneration of rice, and further apply to the selection of bacterial leaf blight-resistant cell lines. Using the mature seeds of Oryza sativa var. Japonica Taken 9 as the explants, the effects of the media, contents of gelrite agar and sucrose and plant growth regulators on callus induction of seeds of O. sativa var. Japonica Taken 9 were evaluated. The results indicated that CS-1 medium containing 2 mg/l 2,4-D, 3% sucrose, 4% gelrite agar at 500 lux light intensity were the best in callus formation, the fresh weight of callus per seed explants about 0.334 g. To observe the effects of 2,4-D contents (0.5-5.0 mg/l) on granular size and texture of callus, granular size would gradually decreased with the increase of 2,4-D content, but callus texture of granular, granulate cluster and massive has been occurred in all treatments. In cell culture, using the callus cultured on CS-1 medium containing 2 mg/l 2,4-D for 45 days as the materials and the suspension cells establish cultured for 10 days could obtained the maximum suspension cells, which about 1.4×106 cells per 3 g fresh weight callus. To evaluate the effects of the fresh weight of callus per explant, plant growth regulator, light intensity and gelrite agar on plantlet regeneration of callus, CS-1 medium containing 2
mg/l NAA, 4 mg/l Kinetin, 3% sucrose and 6% gelrite agar at 3500 lux light intensity had the best result in the rate of plantlet regeneration, which about 60%. Using the suspension cells, induced from the above-mentioned conditions of cell culture, as the materials, Xanthomonas oryzae pv. oryzae as the selection agent and the method of suspension cells co-cultured with X. oryzae pv. oryzae to proceed the selection of bacterial leaf blight-resistant cell lines. The selected bacterial leaf blight-resistant cell lines and culture cells of Oryza sativa var. Japonica Taken 9 were treated with the culture filtrate of X. oryzae pv. oryzae, the survival rate of cells were 96% and 50.4%, respectively. The result indicated that the selected bacterial leaf blight-resistant cell lines were resistant to the culture filtrate of X. oryzae pv. oryzae. The bacterial leaf blight-resistant cell lines subcultured on CS-1 medium containing 2 mg/l 2,4-D could differentiate to clusters, and clusters could differentiate to somatic embryos, but couldn’t continue grew to plantlets.
目錄

中文摘要...................................................Ⅰ
Abstract.................................................Ⅲ
謝誌......................................................Ⅴ
目錄.....................................................Ⅵ
圖表目錄..................................................X
壹、緒言..................................................1
貳、前人研究...............................................4
一、白葉枯病水稻之篩選育種研究概況...........................4
(一)利用傳統抗病育種法進行水稻抗白葉枯病之育種工作............4
(二)利用生物技術育種法進行水稻抗白葉枯病之育種工作............4
二、水稻組織培養之研究概況...................................6
(一)水稻癒傷組織誘導培養...................................6
(二)水稻懸浮細胞培養......................................9
(三)水稻癒傷組織植物再生..................................10
三、水稻白葉枯病研究概況....................................13
(一)水稻白葉枯病之特性....................................13
(二)水稻白葉枯病之病原特異性...............................14
(三)水稻白葉枯病之病徵.....................................15
(四)水稻白葉枯病發生生態...................................16
(五)水稻白葉枯病對水稻生產之影響............................16
(六)抗白葉枯病之抗病性機制其生理生化特性.....................17
(七)防治策略.............................................18
參、材料與方法.............................................19
一、供試材料之製備.........................................19
(一)培養基之製備.........................................19
(二)供試材料種子消毒與組織細胞養...........................20
(三)供試菌株.............................................21
(四)細胞存活率之計算.....................................21
(五)植株再生率之計算......................................22

(六)實驗數據之分析.......................................22
二、水稻台稉9號癒傷組織誘導培養技術之研發......................22
(一)培養基組成對誘導水稻台稉9號種子癒傷組織之影響.............22
(二)培養基內水晶洋菜含量對於誘導水稻台稉9號種子癒傷組織之
影響.................................................22
(三)培養基內蔗糖含量對於誘導水稻台稉9號種子癒傷組織之影響
....................................................23
(四)CS-1培養基內2,4-D含量對誘導台稉9號種子癒傷組織之影響.....23
三、水稻台稉9號細胞懸浮培養技術之開發........................23
(一)癒傷組織誘導時間對水稻台稉9號細胞懸浮培養之影響..........23
(二)以含不同濃度2,4-D誘導之水稻台稉9號種子癒傷組織之細胞
懸浮數..............................................24
四、水稻台稉9號癒傷組織植物再生培養技術之研發.................24
(一)培植體癒傷組織鮮重對水稻台稉9號種子癒傷組織植株再生之
影響................................................24
(二)培養基糖份含量對水稻台稉9號種子癒傷組織植物再生之影響
.....................................................24
(三)CS-1培養基內2,4-D與Kinetin含量對水稻台稉9號種子癒傷
織植物再生之影響.....................................25
(四)CS-1培養基內植物生長調節劑組成對水稻台稉9號種子癒傷組
織植物再生之影響.....................................25
(五)光照度對於誘導水稻台稉9號種子癒傷組織植株再生影響...........26
(六)培養基內水晶洋菜含量對水稻台稉9號種子癒傷組織植株再生
之影響...............................................26
五、抗白葉枯病細胞系之篩選、體胚分化培養與抗性檢測..............26
(一)抗白葉枯病細胞系之篩選.................................27
(二)抗白葉枯病細胞系與白葉枯病菌培養液共同培養之存活細胞.......27
(三)抗白葉枯病細胞系之體胚分化..............................27
(四)抗白葉枯病細胞系對白葉枯病菌培養過濾液之抗性檢測.........28
肆、結果..................................................29
一、水稻台稉9號水稻癒傷組織誘導培養技術之研發.................29
(一)培養基對誘導水稻台稉9號種子癒傷組織之影響...............29
(二)CS-1培養基內水晶洋菜含量對於誘導水稻台稉9號種子癒傷組
織之影響............................................29
(三)CS-1培養基內蔗糖含量對於誘導水稻台稉9號種子癒傷組織之
影響...............................................30
(四)CS-1培養基內2,4-D含量對誘導台稉9號種子癒傷組織之影響...30
二、水稻台稉9號細胞懸浮培養技術之研發.......................31
(一)癒傷組織誘導培養時間對水稻台稉9號細胞懸浮培養之影響.......31
(二)以含不同濃度2,4-D誘導之水稻台稉9號種子癒傷組織之懸浮
細胞數...............................................32
三、水稻台稉9號癒傷組織植株再生培養技術之研發..................32
(一)培植體癒傷組織鮮重量對水稻台稉9號種子癒傷組織植株再生
之影響...............................................32
(二)培養基糖份含量對水稻台稉9號種子癒傷組織植株再生之影響......33
(三)CS-1培養基內2,4-D與Kinetin含量對水稻台稉9號種子癒傷
組織植物再生之影響.....................................33
(四)CS-1培養基內植物生長調節劑組成對水稻台稉9號種子癒傷組
織植株再生之影響......................................34
(五)光照度對誘導水稻台稉9號種子癒傷組織植株再生影響...........34
(六)CS-1培養基水晶洋菜含量對水稻台稉9號種子癒傷組織植株再
生之影響.............................................35
四、抗白葉枯病細胞系之篩選、體胚分化培養與抗性檢測..............36
(一)抗白葉枯病細胞系之篩選.................................36
(二)抗白葉枯病細胞系與白葉枯病菌培養液共同培養之存活細胞.......36
(三)抗白葉枯病細胞系之體胚分化培養..........................36
(四)抗白葉枯病細胞系對白葉枯病菌培養過濾液之抗性檢測..........36
伍、討論..................................................38
參考文獻..................................................45
附錄......................................................89
作者簡介...................................................90
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