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研究生:許馨云
研究生(外文):Hsu Shin-Yun
論文名稱:以白葉枯病菌培養過濾液誘導台稉九號抗病株之篩選
論文名稱(外文):Selection of disease resistant plantlets of Oryza sativa var. Japonica Taiken 9 induced with culture filtrate of Xanthomonas oryzae pv. oryzae
指導教授:鄭秋雄
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:植物保護系所
學門:農業科學學門
學類:植物保護學類
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:105
中文關鍵詞:台稉九號稻白葉枯病菌白葉枯病抗病篩選抗病品系抗病檢測組織培養癒傷組織
外文關鍵詞:Oryza sativa var. Japonica Taiken 9Xanthomonas oryzae pv. oryzaebacterial leaf blightdisease-resistant selectiondisease-resistant plantsresistibility detectiontissue culturecallus
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本研究之目的是利用植物組織培養技術篩選抗白葉枯病水稻品系及研發抗病篩選技術與台稉九號稻癒傷組織之誘導培養技術。抗病篩選技術,則以以100 ml之YPDA液體培養基中加入108 cfu/ml白葉枯病菌並培養72小時之病菌培養過濾液做為篩選劑之篩選效率最佳。以此篩選劑處理小於2 mm(直徑)之癒傷組織,於20 ml之培養過濾液中放入1.5 g癒傷組織,篩選處理6天後,可造成癒傷組織褐變指數約72.83-79.93%。為研發台稉九號稻癒傷組織誘導培養條件,結果以培養於含2 mg/l 2,4-D及2.8 g/l脯胺酸之CS-1培養基,5600-6200 lux光照,培養45天之癒傷組織形成量最佳,單位培植體之癒傷組織鮮重約0.214 g,此法可誘導出球形(nodular callus)硬質之癒傷組織。為篩選出抗白葉枯病水稻品系,以水稻白葉枯病菌培養過濾液為篩選劑,並以培養過濾液之粗汁液連續篩選二次(第一次及第二次篩選)後,再以濃縮1倍之培養過濾液篩選一次(第三次篩選)為篩選方法。結果第一次篩選之抗性癒傷組織系與非抗性癒傷組織系分別為23.21%及76.79%,第二次篩選為15.04%及84.96%,第三次篩選則為4.75%及95.25%。另,第一次篩選之抗性癒傷組織系與非抗性癒傷組織系之植株形成率分別為1.20%及8.17%,第二次篩選為8.24%及0%,第三次篩選則均未再生出植株。取抗性癒傷組織系(褐變級數為0者)再生植株5株、非抗性癒傷組織系再生植株35株(褐變級數為1者)及非抗性癒傷組織系再生植株19株(褐變級數為2及3)及台稉九號稻47株,分別接種白葉枯病菌,結果其抗病品系分別為2株(40%)、14株(48.57%)、5株(26.32%),相對地,台稉九號稻均為感
病。又,取抗病品系自交授粉子代F1(cfXoo-1-1-F1及cfXoo-1-6-F1)與台稉九號稻接種白葉枯病菌,以檢測其抗病遺傳性。結果,其抗病品系分別為8株(42.1%)、15株(35.7%),而台稉九號稻均為感病。
The purposes of this study were to select the bacterial leaf blight- resistant plants of rice, to develop the technologies of disease-resistant selection and callus induction of Oryza sativa var. Japonica Taiken 9 through plant tissue culture. In the technique of disease-resistant selection, added 108 cfu/ml X. oryzae pv. oryzae in 100 ml YPDA liquid medium and the culture filtrate of X. oryzae pv. oryzae cultured for 72 hours as the selection agent provided the best selection result. The callus, little than 2 mm in diameter, were treated with the agent, added 1.5 g callus in 20 ml culture filtrate of X. oryzae pv. oryzae 6 days after selection, which could result in the browning index of callus about 72.83-79.93%. In the culture conditions of the callus induction of Oryza sativa var. Japonica Taiken 9, CS-1 medium containing 2 mg/l 2,4-D and 2.8 g/l proline, at 5600-6200 lux light intensity for 45 days had the best result in callus formation. The fresh weight of callus per seed about 0.214 g,and this method could induced nodular callus. In order to select the bacterial leaf blight-resistant plants of rice, using the culture filtrate of X. oryzae pv. oryzae as the selection agent, and the selection method was to select with the crude culture filtrate of X. oryzae pv. oryzae twice (the first and second selection) and then selected with concentrated
one fold culture filtrate of X. oryzae pv. oryzae once (the third selection). The resistant callus lines and nonresistant callus lines were 23.21% and 76.79% in the first selection, the second selection were 15.04% and 84.96%, and the third selection were 4.75% and 95.25%. The plantlets regeneration rate of the resistant callus lines and nonresistant callus lines were 1.20% and 8.17% in the first selection, the second selection were 8.24% and 0%, and there were not regenerated any plantlets all of them in the third selection. Inoculated 5 plants regenerated from the resistant callus lines (browning level 0), 35 plants (browning level 1) and 19 plants (browning level 2-3) regenerated from the nonresistant callus lines and 47 plants of Oryza sativa var. Japonica Taiken 9 with X. oryzae pv. oryzae, respectively. The disease-resistant plants obtained from these inoculated plants were 2 plants (40%), 14 plants (48.57%) and 5 plants (26.32%), on the contrary, all the plants of Oryza sativa var. Japonica Taiken 9 were sensitivity to X. oryzae pv. oryzae. Inoculated the self-pollinated progeny (cfXoo-1-1 F1 and cfXoo-1-6 F1) of bacterial leaf blight-resistant plants and Oryza sativa var. Japonica Taiken 9 with X. oryzae pv. oryzae to detect the inheritance of resistibility. The disease-resistant plants obtained from these inoculated plants were 8 plants (42.1%), 15 plants (35.7%), and all the plants of Oryza sativa var. Japonica Taiken 9 with X. oryzae pv. oryzae were sensible.
目錄

中文摘要……………………………………………………………………Ⅰ
Abstract……………………………………………………………………...Ⅲ
誌謝………………………………………………………………………….Ⅴ
目錄………………………………………………………………………….Ⅵ
圖表目錄…………………………………………………………………….Ⅹ
壹、緒言……………………………………………………………………...1
貳、前人研究……………………………………………………………….…4
一、水稻白葉枯病…………………………….………………………..…..4
(一)水稻白葉枯病之病原及特異性……………………………………4
(二)水稻白葉枯病之病徵………………………………………………5
(三)白葉枯病之寄主、分布範圍與發生生態…………………………6
(四)水稻白葉枯病對水稻生產之影響………………………………....7
(五)抗白葉枯病之抗病性機制及其生理生化特性……………………8
(六)防治策略……………………………………………………………8
二、抗白葉枯病水稻之篩選育種研究概況……………………………….8
(一)利用傳統抗病育種法進行水稻抗白葉枯病之育種工作…………9
(二)利用生物技術育種法進行水稻抗白葉枯病之育種工作…………9
三、水稻組織培養之研究概況…………………………………………..13
(一)水稻之癒傷組織誘導培養………………………………………..13
(二)水稻之植株再生…………………………………………………..15
參、材料與方法……………………………………………………………..17
一、供試材料之製備……………………………………………………..17
(一)培養基之製備……………………………………………………..17
(二)癒傷組織之誘導培養………………………………………..……17
(三)懸浮細胞之建立培養…………………………….……………….19
(四)水稻無菌植株之培育……………………………………………..19
(五)再生植株之馴化栽培……………………………………………..20
(六)供試對照水稻之培育…………………………….………………..20
(七)供試菌株之採集及分離……………………….…………………..21

(八)癒傷組織褐變級數之評估標準及計算方式……………………..22
(九)細胞存活率之計算………………………………………………..22
(十)植株再生率之計算………………………………………………..22
(十一)植株抗感病程度之評估………………………………………..23
(十二)實驗數據之分析…………………………..…………………….23
二、抗白葉枯病篩選技術之研發………………………………………..23
(一)水稻白葉枯病菌標準曲線之建立………………………………..23
(二)水稻白葉枯病菌之生長曲線……………………………………..23
(三)癒傷組織分別以培養18、36、72及204小時之白葉枯病菌
之培養過濾液處理後之癒傷組織褐變指數……………………24
(四)不同大小癒傷組織經白葉枯病菌培養過濾液處理後癒傷組織
之褐變指數……………………………………………….………24
三、台稉九號稻癒傷組織誘導培養技術之研發……………………….25
(一)CS-1培養基內2,4-D含量對誘導台稉九號稻種子癒傷組織之
影響……………………………………………………………….25
(二)培養基對誘導台稉九號稻種子癒傷組織之影響……………….25
(三)培養基、脯胺酸及酪蛋白水解物對誘導台稉九號稻種子癒傷
組織之影響……………………………………………………….25
(四)CS-1培養基內脯胺酸含量對誘導台稉九號稻種子癒傷組織之
影響……………………………………………………………….26
(五)光照度對於誘導台稉九號稻種子癒傷組織之影響…………...26
四、抗白葉枯病品系之篩選及抗病性檢測……………………..………26
(一)白葉枯病菌培養過濾液對台稉九號稻癒傷組織系褐變之
影響……………………………………………………………….26
(二)白葉枯病菌培養過濾液對台稉九號稻癒傷組織系植株再生之
影響……………………………………………………………….28
(三)經白葉枯病菌培養過濾液篩選之台稉九號稻癒傷組織系再生
植株之抗病性檢測……………………………………………….28
(四)抗白葉枯病品系自交授粉後代F1之抗病性檢測……………...28
(五)抗白葉枯病品系自交授粉子代F1之癒傷組織經白葉枯病菌
培養過濾液處理後之褐變指數……………………………….…28
(六)抗白葉枯病品系自交授粉子代F1之培養細胞經白葉枯病菌
1倍濃縮培養過濾液處理後之細胞存活率…………………….29
肆、結果……………………………………………………………………..30
一、抗白葉枯病篩選技術之研發……………………………………….30
(一)水稻白葉枯病菌標準曲線之建立……………………………….30
(二)水稻白葉枯病菌之生長曲線……………………………………..30
(三)癒傷組織分別以培養18、36、72及204小時之白葉枯病菌
之培養過濾液處理後之癒傷組織褐變指數…………………….30
(四)不同大小癒傷組織經白葉枯病菌培養過濾液處理後癒傷組織
之褐變指數………………………………………………...……..31
二、台稉九號稻癒傷組織誘導培養技術之研發……………………….31
(一)CS-1培養基內2,4-D含量對誘導台稉九號稻種子癒傷組織之
影響……………………………………………………………….31
(二)培養基對誘導台稉九號稻種子癒傷組織之影響………………..32
(三)培養基、脯胺酸及酪蛋白水解物對誘導台稉九號稻種子癒傷
組織之影響……………………………………………………….32
(四)CS-1培養基內脯胺酸含量對誘導台稉九號稻種子癒傷組織之
影響………………………………………………...……………..33
(五)光照度對於誘導台稉九號稻種子癒傷組織之影響……………..34
三、抗白葉枯病品系之篩選及抗病性檢測…………………………….34
(一)白葉枯病菌培養過濾液對台稉九號稻癒傷組織系褐變之
影響……………………………………………………………...34
(二)白葉枯病菌培養過濾液對台稉九號稻癒傷組織系植株再生之
影響……………………………………………………………….35
(三)經白葉枯病菌培養過濾液篩選之台稉九號稻癒傷組織系再生
植株之抗病性檢測…………………………………...………….35
(四)抗白葉枯病品系自交受粉後代F1之抗病性檢測……………...36
(五)抗白葉枯病品系自交授粉子代F1之癒傷組織經白葉枯病菌
培養過濾液處理後之褐變指數……………………………….36
(六)抗白葉枯病品系自交授粉子代F1之培養細胞經白葉枯病菌
1倍濃縮培養過濾液處理後之細胞存活率……………………...37
伍、討論……………………………………………………………………..38
參考文獻………………………………………………………………….....47
附錄………………………………………………………………………….91
作者簡介…………………………………………………………….…....105
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