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研究生:周汝潔
研究生(外文):Ju-Chieh Chou
論文名稱:繼代培養及植物生長調節劑對丹參癒合組織二萜類生合成含量之影響
論文名稱(外文):Influence of Age and Plant Growth Regulators on Diterpenoids Contents in Callus of Salvia miltiorrhiza Bunge
指導教授:蔡新聲蔡新聲引用關係古新梅
指導教授(外文):Hsin-Sheng TsayHsin-Mei Ku
口試委員:張宏祺
口試委員(外文):Hung-Chi Chang
口試日期:2016-07-27
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農藝學系所
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:54
中文關鍵詞:丹參癒合組織二萜類繼代培養玉米素玉米素核苷
外文關鍵詞:Salvia miltiorrhizacallusditerpenoidsplant tissue agezeatinzeatin riboside
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丹參(Salvia miltiorrhiza Bunge)為脣形科(Labiatae)鼠尾草屬,多年生草本藥用植物,主要以根部入藥,具有祛瘀止痛、活血通經、養血安神等作用。其根部富含二萜類可預防及治療心血管疾病,是為我國常用的活血化瘀之藥,現代臨床上則多做為治療心血管疾病之用。過去研究已知丹參癒合組織處理1 mg/L trans-zeatin roboside (ZR)時會轉變為磚紅色,並累積大量的丹參酮I、丹參酮IIA及隱丹參酮,而本論文將探討繼代培養超過十年的舊丹參癒合組織以及繼代培養6個月的新丹參癒合組織處理ZR時二萜類含量之差異;丹參癒合組織生產二萜類時處理ZR的最適濃度,以及是否可以zeatin取代ZR誘導丹參癒合組織大量生產二萜類。結果顯示舊丹參癒合組織質地脆弱易碎、顏色透明、含水量偏高,且無法透過處理ZR誘導二萜類的生合成;而新丹參癒合組織質地較硬不易碎、顏色飽滿呈淡黃色、含水量較低,處理ZR後顏色轉變為磚紅色,並可誘導二萜類大量生合成,於處理15至20天開始有顯著差異,處理30天時達到最高量。以1 mg/L ZR誘導丹參癒合組織可獲得最高含量的丹參酮I及隱丹參酮, 以2 mg/L ZR誘導可獲得最高含量的丹參酮IIA,但處理1 mg/L ZR可獲得最高總二萜類的含量。而以zeatin取代ZR亦可誘導丹參癒合組織大量生合成二萜類,且可合成更高量之丹參酮I及隱丹參酮,並可有效降低生產成本。

Salvia miltiorrhiza (Danshen), a well-known traditional Chinese medicinal herb belongs to family Lamiaceae. Roots of S. miltiorrhiza contain abundant diterpenoid quinones compounds. These bioactive compounds are widely used for the prevention and treatment of cardiovascular and other diseases. Based on the previous study, it is known that S. miltiorrhiza callus turns brick red in color when treated with 1 mg/L trans-zeatin roboside (ZR) and accumulates abundant diterpenoid quinones compounds like tanshinone I, tanshinone IIA, and cryptotanshinone. The present study was carried out to understand the influence of age on contents of diterpenoids in callus of S. miltiorrhiza. We analyzed the diterpenoids contents the old callus (cultured for over 10 years) and new callus (cultured for less than 1 year). HPLC analysis results showed that compared to the new callus, old callus when treated with ZR could not synthesize diterpenoids, and the callus became friable, transparent and had higher water content. On the other hand, new callus when treated with ZR became brick red in color and synthesized abundant diterpenoids. The highest amounts of all three diterpenoids was recorded at 30 days of culture and cryptotanshinone content in the callus was higher compared to tanshinone I, tanshinone IIA. To understand the the influence of plant growth regulators on diterpenoids contents in callus of S. miltiorrhiza, we tested different concentrations of ZR (0.1, 0.5, 1, 2 and 4 mg/L). The results showed that the highest amounts for tanshinone IIA and cryptotanshinone were obtained on the medium with 1 mg/L ZR, while the maximum tanshinone I content was recorded on the medium with 2 mg/L ZR. Also, an experiment was carried out to test if ZR could be replaced in the medium with zeatin since the later is less expensive. It was observed that S. miltiorrhiza callus cultured on medium with zeatin also become brick red in color and synthesized higher amounts of tanshinone I and cryptotanshinone compared to medium with ZR. These results could be helpful in producing the bioactive compounds in S. miltiorrhiza by using tissue culture technique though out the year and without sacrificing the S. miltiorrhiza plants.

中文摘要......................................i
Abstract......................................ii
目錄......................................iii
略字表......................................v
表目錄......................................vi
圖目錄......................................vii
第一章 前言......................................1
第二章 前人研究......................................3
一、丹參之簡介......................................3
(一) 丹參植株型態......................................3
(二) 丹參產地與分布......................................3
(三) 丹參藥理作用......................................3
(四) 丹參活性成分......................................4
二、利用癒合組織生產二次代謝物......................................6
三、玉米素之介紹......................................8
(一) 玉米素的發現......................................8
(二) 玉米素於植物組織培養的利用......................................9
四、利用組織培養方法生產丹參活性成分......................................9
第三章 材料與方法......................................11
一、試驗材料......................................11
二、丹參癒合組織大量繁殖......................................11
(一) 誘導野生型丹參癒合組織......................................11
(二) 癒合組織之繼代培養......................................11
三、誘導丹參二萜類生合成......................................12
(一) 誘導二萜類生合成培養基......................................12
(二) 誘導丹參二萜類生合成試驗......................................12
四、丹參二萜類含量測定......................................13
(一) 成份測定使用儀器......................................13
(二) 標準品製備......................................13
(三) 丹參檢品製備......................................13
(四) 高效液相層析法......................................14
五、資料統計與分析......................................14
第四章 結果......................................15
一、兩種丹參癒合組織外觀及含水量測定......................................15
二、丹參癒合組織處理ZR外觀型態與成分分析......................................15
三、丹參癒合組織處理不同濃度ZR外觀型態與成分分析......................................16
四、丹參癒合組織處理zeatin外觀型態與成分分析......................................17
第五章 討論......................................19
一、兩種丹參癒合組織外觀及含水量......................................19
二、丹參癒合組織處理ZR外觀型態與成分分析......................................19
三、丹參癒合組織處理不同濃度ZR外觀型態與成分分析......................................20
四、丹參癒合組織處理zeatin外觀型態與成分分析......................................20
參考文獻......................................36


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