臺灣博碩士論文加值系統

北板藍（Bei-Ban-Lan, Isatis indigotica），又稱為菘藍，屬十字花科（Cruciferae）二年生草本藥用植物。其乾燥根是中藥板藍根之基原材料，台灣目前尚無自產，現今藥材皆仰賴大陸進口。但由於來源及產地不同，使得藥材基源及品質不一。本論文的目的，在探討北板藍植物組織培養條件，作為日後種苗大量繁殖及利用懸浮培養生產藥用成分之基礎研究。
以苗齡三-四週無菌播種北板藍實生苗之根部、下胚軸及子葉為培植體，三種培植體中以子葉對癒合組織誘導效果最佳。培養於含有0.5 mg/L BA、0.5 mg/L 2,4-D及3﹪蔗糖之MS培養基中，經暗室培養一個月，癒合組織誘導率達1000%。癒合組織繼代培養於含0.5 mg/L BA、0.5 mg/L 2,4-D及3﹪蔗糖之MS培養基中，經暗室培養一個月，其鮮、乾重量較1/2 MS、B5或LS基本培養基為高，其最適合北板藍癒合組織增殖。BA、KIN及TDZ三種細胞分裂素，均可誘導癒合組織枝系器官發生，其中以3.0 mg/L TDZ之誘導率可達1000%。以上胚軸以上枝系為培植體培養於含1.0 mg/L BA處理對北板藍叢生枝誘導的效果最佳，可達53 (枝/培植體)。以根部、下胚軸及子葉為培植體，皆可在含有BA之MS培養基中，直接誘導不定芽形成，其中以下胚軸於3.0 mg/L BA中，直接不定芽發生數為213芽/培植體最佳。根部器官發生則以北板藍上胚軸以上枝系或叢生枝誘導所得之枝系培養於含0.1 mg/L NAA之MS培養基中，可獲得82根/培植體為最佳。
玻璃質化(hyperhydricity)是北板藍進行組織培養常遭遇的問題，本試驗結果認為高濃度(1.0 mg/L)的細胞分裂素，尤其是BA，是造成北板藍組織培養玻璃質化重要因素之一。增加試管內外通氣性，則可有效降低玻璃質化發生程度。因此推測組培容器內相對溼度或乙烯濃度也是影響北板藍玻璃質化發生的原因。

Ban-Lan-Gen（Isatis indigotica）,genus of Isatis in the family of Crucifera, and it is a biennial herb, other names is bei-ban-lan. The purpose of this study was to establish the tissue culture techniques for mass propagation of Isatis indigotica ,and followed with cell suspension culture to produce the secondary metabolites. The results are summarized as follows：
1.Seeds of Isatis indigotica were germinated on the medium containing 1/10 MS basal salts,1% sucrose and 0.7% agar（pH＝6.0）, after two weeks under dark, the optimum germination rate was 855%.2.The explants of root tips、hypocotyls and cotyledons of seedling were used for callus induction on the MS basal medium containing BA（0,0.5,1,2,3,5 mg/L）and 2,4-D（0,0.5,1,2,3,5 mg/L）.The results showed that 1000% explants form callus after 30 days under dark on the MS basal medium containing 0.5 mg/L BA and 0.5 mg/L 2,4-D and 3% sucrose, and the cotyledons were the best explant than root tips and hypotyls for callus induction.3.The best basal salts medium for callus proliferation was MS medium, after 30 days under dark ,the fresh and dry weight of callus were better than on the 1/2MS、B5 and LS basal salts medium.4.The callus on the MS medium containing 3.0 mg/L TDZ、3% sucrose and 0.7%agar（pH＝6.0）,after 30 days under light, the shoots formation rate was 1000%.5.The hypercotyls on the MS medium containing 1.0 mg/L BA、3% sucrose and 0.7%agar（pH＝6.0）can induce the mass adventitious shoots formation 53 shoots per explant. 6.Adventitious buds can be induced from hypocotyls when cultured on the MS medium containing 3.0 mg/L BA , 3% sucrose and 0.7%agar（pH＝6.0）medium containing, the regeneration rate was 213 buds per explant than root tips and cotyledon.7.Roots can be induced from hyperotyls when cultured on the MS medium containing 0.1 mg/L NAA, 3% sucrose and 0.7%agar（pH＝6.0）,the results were 82 roots per explant than other condition.8.We used gas-permeable surgical tape﹙Milipore 3M﹚ for sealing tube in this test, it can reduce the relative humidity in the vessels, and no hyperhydricity shoots formation.

中文摘要………………………………………………………………………….……i
英文摘要……………………………………………………………………………....ii
總目錄………………………………………………………………………………...iv
表目錄………………………………………………………………………………..vii
圖目錄……………………………………………………………………………….viii
縮寫…………………………………………………………………………………...ix
第一章 前言…………………………………………………………………………..1
第二章 文獻探討……………………………………………………………………..3
2.1板藍根之本草考察……………………………………………………………..3
2.2板藍根藥用植物學及生藥學文獻考察……………………..…………………4
2.3板藍根之成份及藥理作用文獻考察………………………..…………………5
2. 4 北板藍及十字花科植物組織培養文獻回顧….………..………………………5
2.4.1培植體種類與年齡………………………………………………………...6
2.4.2培養基種類及組成………………………………………………………...6
2.5植物玻璃質化……………………………………………………..……………8
2.5.1細胞分裂素、培養基凝膠物質的種類及濃度..………………….…………8
2.5.2培養容器中的溫度及相對溼度..…..………….……………………………..9
第三章 材料及方法....................................................................................................10
3.1植物材料............................................................................................................10
3.2試驗方法............................................................................................................10
3.2.1培養基成分與製備.......................................................................................10
3.2.2培養環境.......................................................................................................11
3.2.3癒合組織乾、鮮重測量.................................................................................11
3.2.4觀察記錄與統計分析法………..........................................................……..11
3.2.5北板藍無菌播種...........................................................................................11
3.3 北板藍組織培養...............................................................................................12
3.3.1間接枝系發生...............................................................................................12
3.3.1.1培植體種類對北板藍癒合組織誘導率之影響......................................12
3.3.1.2不同基本鹽類培養基對北板藍癒合組織繼代增殖之影響..................12
3.3.1.3不同細胞分裂素對北板藍癒合枝系器官發生之影響..........................12
3.3.2直接枝系發生...............................................................................................13
3.3.2.1培植體種類及BA濃度對北板藍培植體枝系直接發生之影響...........13
3.3.3叢生枝誘導...................................................................................................13
3.3.3.1不同細胞分裂素對北板藍叢生枝誘導之影響......................................13
3.3.4根器官發生...................................................................................................13
3.3.4.1生長素濃度對北板藍根系誘導之影響..................................................13
3.3.5組織培養再生植株出瓶馴化.......................................................................13
3.4 玻璃質化原因探討與解決之道.......................................................................14
3.4.1植株玻璃質化改善試驗...............................................................................14
第四章 結果........................................................................................................15
4.1間接枝系發生....................................................................................................15
4.1.1 BA及2,4-D對北板藍培植體癒合組織誘導之影響.................…….........15
4.1.2不同培養基基本鹽類對北板藍癒合組織繼代增殖之影響............……...15
4.1.3.細胞分裂素對北板藍癒合組織枝系形成之影響...........................……....16
4.2直接枝系發生....................................................................................................16
4.2.1 BA濃度對北板藍不同培植體器官直接發生之影響………………….....16
4.3叢生枝誘導........................................................................................................16
4.3.1 BA濃度對北板藍上胚軸叢生枝系形成之影響....................…...……......16
4.4根器官發生........................................................................................................17
4.4.1 NAA濃度對北板藍上胚軸培植體根系發生之影響………….........…….17
4.5玻璃質化原因探討及解決之道........................................................................17
4.5.1BA及鋁箔封口對北板藍玻璃質化苗之影響....................................……..17
第五章 討論................................................................................................................18
5.1北板藍癒合組織誘導及生長試驗....................................................................18
5.2直接器官發生....................................................................................................19
5.3間接枝系器官發生...............................................................................…….........19
5.4北板藍叢生枝誘導及發根試驗........................................................................20
5.5植株玻璃質化....................................................................................................21
第六章結論..................................................................................................................23
參考文獻……………………………………………………………………………….43
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