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研究生:謝育玲
研究生(外文):YuLing Hsieh
論文名稱:以樟樹及相思樹枝梢材培養牛樟芝:初期生長及三萜類成分分析
論文名稱(外文):The Primary Growth and Triterpene Analysis of Antrodia cinnamomea Cultured on Branch-Based Substrates of Cinnamomum camphora (L.) Presl. and Acacia confusa Merr.
指導教授:廖宇賡李世豪李世豪引用關係
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:森林暨自然資源學系研究所
學門:農業科學學門
學類:林業學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
中文關鍵詞:牛樟芝扣子體栽培介質菌絲體原基三萜類
外文關鍵詞:Antrodia cinnamomeaclampculture substratemyceliaprimordiatriterpene
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本研究以非牛樟木介質培養牛樟芝(Antrodia cinnamomea T T Chang &W N Chou),來避免牛樟樹(Cinnamommum kanehirai Hay.; Ck)因過度伐採而瀕臨絕種。所使用非牛樟木材料有樟樹(C. camphora (L.) Presl. ; Cc)及相思樹(Acacia confusa Merr.; Ac),並以牛樟樹為對照。此三種樹材皆取自立木枝梢材,經去皮後,製成小徑級椴木(直徑<7 cm)、木塊(2 × 2 × 2 cm3)及木粉調製之仿木塊(6 × 6 × 6 cm3)等介質形態。牛樟芝菌絲接種後初期生長顯示,在Cc或Ac的三種不同形態介質中均生長良好,椴木中菌絲經8週培養可佈滿介質表面;在木塊中菌絲的生長則提早在第4週完全覆蓋木塊培養基,且不因樹種的不同而有所差異。仿木塊試驗則以Ck木粉製成之介質菌絲生長較快,接種8週後完全覆滿,但同一時間,Cc與Ac仿木塊上菌絲覆蓋率僅達80%。另仿木塊介質中菌絲經4個月培養後均有原基產生。本研究牛樟芝菌絲體之接種源以平板培養基培養8週後,在菌落外圍可觀察到扣子體構造,證明此階段已有生殖菌絲,並具備形成子實體的條件。以HPLC分析三種樹種木粉介質與其所培育牛樟芝萃取物之三萜類成分,顯示Ck、Cc與Ac皆無牛樟芝之三萜類成分,所培養之牛樟芝可由自行合成部分羊毛甾烷型三萜類:dehydroeburicoic acid、15α-acetyl dehydrosulphurenic acid、3β, 15α-dihydroxy lanosta-21-oic acid、dehydrosulphurenic acid以及sulphurenic acid,但仍無子實體特有麥角甾烷型之三萜類。本研究所培養之牛樟芝雖經過9個月培養,僅獲得子實體原基,並經三萜類成分分析,鑑定其為菌絲體轉變成子實體之過渡階段,以此系統培養牛樟芝9個月,尚不足以形成牛樟芝子實體。
This study deals with culturing Antrodia cinnamomea TT Chang &WN Chou in vitro by using wood material of Cinnamomum camphora (L.) Presl. (Cc) and Acacia confuse Merr. (Ac), collected from debarked branches of living trees as incubation substrate. The same collection of C. kanehirae Hay. (Ck) was also included as control. These three wood materials were processed into small logs (with diameter less than 7.0 cm), blocks (2 × 2 × 2 cm3) and fine chips to be further shaped into cubic cakes (6 × 6 × 6 cm3). Preliminary observations showed that the hypha of A. cinnamomea grew well on Cc and Ac substrates (block medium and logs) and simultaneously covered the substrate surface within 4 or 8 weeks, respectively. As compared with fully (100%) hyphal coverage on Ck cake (control), hyphal growth on Cc and Ac cakes was slower, with 80% surface coverage. The primordia of A. cinnamomea was observed on shaped cakes for 4-month culture. The clamp cells were also observed on the A. cinnamomea hypha (the inoculant) incubated on medium plate for 8 weeks. This evidence revealed that the inoculant cultures were developing into generative hypha, and capable of forming fruiting body. The HPLC analysis of wood powder and A. cinnamomea extraction demonstrated that the wood material of all species contain no triterpenes specifically determined in A. cinnamomea. The A. cinnamomea and its primordia revealed that the bio-synthesis of lanostane-type triterpenes, like dehydroeburicoic acid, 15α-acetyl dehydrosulphurenic acid, 3β, 15α-dihydroxy lanosta-21-oic acid, dehydrosulphurenic acid and sulphurenic acid, was independent of culture substrates. Although the primordia was obtained from 9-month-old cultures, its growth stage was identified as in a transition status prior to the fruiting body formation due to the absence of fruiting-body ergostane-type triterpenes. The culture system presented in this study does not fully fill the requirement of forming fruiting body during the 9-month culture period.
I、前言 1
Ⅱ、前人研究 2
(Ⅰ)牛樟芝的簡介 2
(Ⅱ) 牛樟芝的生理活性成分 4
(Ⅲ) 牛樟芝藥理上的應用 8
(Ⅳ)牛樟芝子實體的鑑定 10
(Ⅴ) 牛樟芝人工栽培方法 15
Ⅲ、材料與方法 18
(I) 菌種來源 18
(II) 培養介質之製備 18
(III) 接種 20
(Ⅳ) 牛樟芝菌絲的顯微觀察 20
(Ⅴ) 三萜類成分分析 20
Ⅳ、結果 23
(I) 不同木材介質接種牛樟芝生長狀況 23
(II) 牛樟芝菌絲的顯微觀察 31
(III) 原基的發生 33
(Ⅳ) 化學分析結果 36
Ⅴ、討論 44
Ⅵ、參考文獻 51

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