臺灣博碩士論文加值系統

絞股藍（Gynostemma pentaphyllum (Thunb.) Makino）為葫蘆科（Cucurbitaceae）絞股藍屬多年生宿根性蔓生草本，因其含有近九十種與人參皂甙結構類似之化合物而深受矚目。
本研究以農桿根群菌（Agrobacterium rhizogenes）ATCC 15834對絞股藍葉片外植體進行感染，並成功誘導產生毛狀根。經PCR確認後，選取生長快速、分支多之毛狀根系進行單株化。比較7株毛狀根單株在不同基礎培養基之生長狀況及絞股藍皂甙產量之差異，最後選定高產性毛狀根系GP-01。以MS為基礎培養基，探討毛狀根生長與二次代謝物累積之動態變化，培養至49天時，毛狀根乾重7.3 g L-1，皂甙含量為乾重之3.8%，總皂甙生產之動態變化類型呈明顯的”生長相關”特性，總皂甙產量最高可達280 mg L-1。毛狀根培養過程中並無絞股藍皂甙釋放至培養基。
激發因子可以誘導植物產生植物防禦素（phytoalexins），因此成為提高植物毛狀根之二次代謝物產量的一種方法。本研究探討不同來源激發因子對絞股藍毛狀根生長及皂甙生合成之影響。實驗結果顯示，激發因子之作用效果與激發因子之種類、濃度、作用時間及毛狀根之生長狀態有關。以茉莉酸甲酯（MJ）的誘導效果最好，最適作用濃度為200 μM。毛狀根在對數生長末期對MJ之作用最敏感。在加入200 μM MJ 3天後收穫，毛狀根中皂甙之含量達4.5% DW，比對照組（3.7% DW）提高21%。
針對基礎培養基內各主要成分對絞股藍毛狀根生長及皂甙生合成之影響進行了研究，確定了生長（growth medium, GM）及生產（production medium, PM）培養基配方。絞股藍毛狀根先以GM培養基培養38天，繼之以PM培養基繼續培養18天後收穫。證實以二階段培養方式確實可有效的促進絞股藍皂甙之生產，使總皂甙產量比GM一階段培養增加126%。
欲利用生物反應器進行毛狀根高密度培養時，如何實現毛狀根接種的自動化與均勻化以及如何有效提高反應器之空間利用率將是生物反應器實用化的關鍵因素。本研究利用均質機將毛狀根切成適當長短，再接種入反應器。不僅操作手續簡便、製程放大容易，同時在反應器中設置不�袗�網作為毛狀根生長之支撐介質，使毛狀根能充分利用反應器空間生長，達成高密度培養之目的。比較絞股藍毛狀根在三種不同型式生物反應器：氣泡塔式反應器（bubble column）、改良的機械攪拌式反應器（modified stirred tank without impeller）及機械攪拌式反應器（stirred tank with impeller）之生長及皂甙生合成狀況。結果發現毛狀根在氣泡塔式反應器及改良的機械攪拌式反應器中之生長速率及皂甙含量均優於機械攪拌式反應器，同時毛狀根在氣泡塔式反應器及改良的機械攪拌式反應器中之生長分佈亦比機械攪拌式反應器均勻。絞股藍毛狀根之擴大培養在改良的機械攪拌式反應器得到較好之結果，以MS為基礎培養基在25℃培養50天後，得到生物量乾重為7.1 g L-1，與三角瓶實驗結果相差無幾。而總皂甙含量4.7%DW，則明顯優於三角瓶實驗結果。
本研究從絞股藍毛狀根培養獲得遠比原植物根部含量高出許多之絞股藍皂甙，證實毛狀根具有合成與原植物相同或類似水準之二次代謝物的能力。雖然絞股藍毛狀根之皂甙含量低於市售品10~30%，但傳統生產方式一年只有1~2穫，且受自然環境及栽培條件等之影響，品質難以均一。與傳統栽培方式相比，利用反應器進行毛狀根擴大培養則可全年生產，因此具有很大的發展潛力。
本研究證實，利用毛狀根培養生產二次代謝產物，提供了可靠、全年生產、不受地區、緯度、氣候、季節、土壤污染、病蟲害的威脅以及政治因素之影響。生產製程的主控權完全由培養者掌控，培養者可以透過毛狀根系的選擇及培養基的最適化來提高產率，除了降低生產成本外，也能提高產品品質。因此，利用毛狀根培養生產高價值之植物二次代謝產物（如絞股藍皂甙）未來具有很大的發展潛力。

Gynostemma pentaphyllum (Thunb.) Makino, which belongs to the Cucurbitaceae family, is a perennial, deciduous, creeping herb. Phytochemical studies of G. pentaphyllum have identified roughly 90 dammarane-type saponin glycosides known as gypenosides which closely resembles the ginsenosides found in Panax ginseng.
Hairy root cultures of G. pentaphyllum were established by infecting leaf discs with Agrobacterium rhizogenes ATCC 15834. Seven transformed root clones were established and examined for their growth rate and gypenoside productivity in various basal media. Clone GP-01 was selected from 7 hairy root clones based on its gypenosides productivity and stability. Kinetic studies revealed the dry biomass of the hairy roots in MS medium was 7.3 g L-1 and the gypenoside content was 3.8 % DW in a 49 days’ culture. The pattern of gypenoside accumulation was “growth–associated” with a peak value of 280 mg L-1. No gypenosides were released into the medium during the cultures.
Elicitor can be used to improve the production of secondary metabolites in hairy root cultures because of its ability to induced phytoalexins in plant. To investigate the effects of different elicitors on gypenoside production in hairy root cultures of G. pentaphyllum. The results showed that elicitation was dependent on the elicitor type, concentration, the incubation period and the physiological stage at which the hairy root cultures were treated. Among these three elicitors, 200 μM MJ had the highest inducing efficiency at the late exponential growth stage. The gypenoside content of the hairy root cultures treated with MJ elicitor was 4.5% DW, which was 21% higher than the control (3.7% DW).
For the enhancement of gypenoside production by G. pentaphyllum hairy roots, the effects of medium constituents on hairy root cultures were investigated in flasks. Based on these results, a step-wise medium shift strategy was developed in which GM (growth medium) was used for cultivation for the first 38 days and PM (production medium) for the remainder of the cultivation period. The gypenoside production obtained by the strategy was 586 mg L-1, which was 126% higher than those in the single medium (GM).
Two areas that have been identified as key factors for large-scale implementation are: the development of suitable inoculation techniques and strategies to provide uniform root distribution within bioreactors. In this study, inoculation of large-scale hairy root culture reactors can be carried out by briefly homogenizing bulk root tissue, followed by aseptic transfer as a slurry to the reactor. Uniform root distribution can be achieved in bioreactors by entrapment of the growing root inoculum onto stainless steel mesh in gas-phase reactors operation. Hairy root cultures of G. pentaphyllum were cultivated in three different culture systems: a bubble column, a modified stirred tank (without impeller) and a stirred tank (with impeller). The growth rate and gypenoside contents of hairy root cultures in the bubble column and the modified stirred tank were higher than that in the stirred tank. The hairy root distribution in the bubble column and the modified stirred tank were also better than in the stirred tank. The roots grown in modified stirred tank reactor showed the best performance in terms of dry weight (7.1 g L-1) and gypenoside content (4.7 g L-1), the latter of which was better than that of shake-flask data.
In this study high levels of gypenosides were obtained from hairy roots despite distribution results showing relatively low gypenoside levels in native roots of G. pentaphyllum. This study demonstrated that transformed roots can synthesize and store significant quantities of secondary metabolites. Although the hairy roots under these conditions produced approximately 10% to 30% less gypenosides than commercial sources of G. pentaphyllum, the growing time was much shorter when compared to field-grown plants.
Therefore, in this study, we demonstrate the induction of hairy roots and the establishment of the culture followed by the infection with A. rhizogenes to grow more rapidly and to produce the gypenosides more efficiently than the ordinary field-grown cultivars. With hairy root cultures, product quality and quantity are easy to control because natural variances in seasonal climates and geographical environments are excluded and culture conditions and process variables are easily optimized. The hairy root cultures have been considered as a potential alternative for production of gypenosides.

目 錄
頁次
誌謝 ---------------------------------------------- i
中文摘要 ------------------------------------------ ii
英文摘要 ------------------------------------------ iv
目錄 ---------------------------------------------- I
圖目錄 -------------------------------------------- V
表目錄 -------------------------------------------- VIII

第一章 緒論 --------------------------------------- 001
壹、前言 ------------------------------------------ 001
貳、研究目的 -------------------------------------- 003
參、研究內容 -------------------------------------- 006

第二章 絞股藍毛狀根懸浮培養系統之建立 ------------- 008
壹、文獻探討 -------------------------------------- 008
一、毛狀根形成之機制 ------------------------------ 008
1. 農桿根群菌致病結構及功能 ----------------------- 008
2. 農桿根群菌感染機制 ----------------------------- 012
3. 毛狀根的建立與培養 ----------------------------- 013
4. 毛狀根之鑑定 ----------------------------------- 017
5. 影響轉殖效率的因素 ----------------------------- 018
6. 提高轉殖效率之方法 ----------------------------- 020
7. 毛狀根的特性 ----------------------------------- 020
8. 藥用植物毛狀根之應用 --------------------------- 020
二、絞股藍之研究與利用 ---------------------------- 021
1. 絞股藍之本草考證 ------------------------------- 021
2. 型態與特徵 ------------------------------------- 023
3. 植物資源 --------------------------------------- 025
4. 絞股藍之化學組成 ------------------------------- 025
5. 絞股藍之藥理作用 ------------------------------- 027
三、研究目的 -------------------------------------- 028
貳、材料與方法 ------------------------------------ 030
一、材料與藥品 ------------------------------------ 030
二、實驗方法 -------------------------------------- 032
參、結果與討論 ------------------------------------ 040
1. 毛狀根之誘導與培養 ----------------------------- 040
2. 感染之確認 ------------------------------------- 041
3. 高產性毛狀根系之篩選 --------------------------- 045
4. 最適接種量 ------------------------------------- 045
5. 絞股藍毛狀根生長動力學之探討 ------------------- 049
6. 薄層層析與高效液相層析 ------------------------- 060
肆、結論 ------------------------------------------ 066

第三章 二次代謝產物之增產策略 --------------------- 067
第一部份 激發因子的影響 --------------------------- 069
壹、前言 ------------------------------------------ 069
1. 激發因子（elicitor）的定義及分類 --------------- 069
2. 激發因子的作用機制 ----------------------------- 070
3. 真菌激發因子 ----------------------------------- 072
4. 水楊酸與茉莉酸 --------------------------------- 072
貳、材料與方法 ------------------------------------ 074
一、材料與藥品 ------------------------------------ 074
二、實驗方法 -------------------------------------- 075
參、結果與討論 ------------------------------------ 077
1. 不同種類及濃度之激發因子的影響 ----------------- 077
2. MJ作用時間的影響 ------------------------- 082
3. 植物細胞生理狀態之影響 ------------------------- 086
肆、結論 ------------------------------------------ 088
第二部份 培養基組成之改良及二階段培養 ------------- 090
壹、前言 ------------------------------------------ 090
1. 基礎培養基之種類 ------------------------------- 090
2. 碳源 ------------------------------------------- 091
3. 氮源 ------------------------------------------- 092
4. 鉀離子 ----------------------------------------- 092
5. 磷酸鹽 ----------------------------------------- 093
6. pH值 ------------------------------------- 093
7. 有機添加物 ------------------------------------- 094
8. 二階段培養 ------------------------------------- 094
貳、材料與方法 ------------------------------------ 095
一、材料與藥品 ------------------------------------ 095
二、實驗方法 -------------------------------------- 095
參、結果與討論 ------------------------------------ 098
一、培養基之改良 ---------------------------------- 098
1. 基礎培養基之選擇 ------------------------------- 098
2. 碳源之影響 ------------------------------------- 100
3. 氮源之影響 ------------------------------------- 105
4. 鉀離子之影響 ----------------------------------- 112
5. 磷酸鹽的影響 ----------------------------------- 113
6. 起始pH之影響 ----------------------------------- 113
7. 有機添加物之影響 ------------------------------- 120
二、二階段培養方式 -------------------------------- 123
肆、結論 ------------------------------------------ 132

第四章 擴大培養條件探討 --------------------------- 133
壹、前言 ------------------------------------------ 133
1. 毛狀根培養特性 --------------------------------- 133
2. 毛狀根培養生物反應器之主要類型及特點 ----------- 136
貳、材料與方法 ------------------------------------ 143
一、材料與藥品 ------------------------------------ 143
二、實驗方法 -------------------------------------- 143
參、結果與討論 ------------------------------------ 146
1. 接種方式之影響 --------------------------------- 146
2. 不同通氣量之影響 ------------------------------- 147
3. 不同型式反應器之影響 --------------------------- 147
4. 二階段培養測試 --------------------------------- 152
肆、結論 ------------------------------------------ 155
第五章 總結 --------------------------------------- 156
第六章 未來展望 ----------------------------------- 159
第七章 參考文獻 ----------------------------------- 160

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