丹參組織培養苗馴化處理及其植株丹參酮含量分析
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(2) 22. 台灣農業研究. 第 56 卷. 第 1 期. 參市場所需藥材皆仰賴中國進口，近年來其野生資源逐漸減少，雖可利用種子，分根、蘆頭或扦插 等方法進行丹參種苗繁殖栽培，然各地藥材品質多參差不齊，更有偽劣品充斥市場的現象 (Guo et al. 2002)。利用植物組織培養技術不僅可縮短繁殖與栽培時間，生產之種苗更具有一致性的優點， 而且組織培養技術於人為環境的控制下，可進一步運用科學方法提高代謝物之品質和產量 (Song et al. 1998)。Chen et al. (2005) 已成功利用組織培養技術大量繁殖丹參組培芽體；但組培芽體發根及 組培苗馴化的方法則未見報導。本研究利用吲哆丁酸與改善培養容器封口以促進丹參組培芽體發根 與組培苗馴化存活率，並藉由 HPLC 方法檢測及比較組培苗田間栽種植株與市售丹參藥材之丹參 酮含量，評估臺灣利用組培技術生產大量種苗用以自產丹參藥材的可行性。. 材料與方法 供試材料與培養基 本研究採用之丹參 (Salvia miltiorrhiza) 植株由中國醫藥大學陳忠川教授提供、鑑定，栽培於 行政院農委會農業試驗所。依據 Chen et al. (2005) 方法，利用丹參莖節腋芽作為培殖體，培養於 含有適量苄氨基嘌呤 (6-benzyladenine; BA) 與奈乙酸 (α-naphthileneacetic acid ; NAA) 之 MS 培養 基中，可成功地誘導叢生芽體形成及建立初代無菌組培芽體以作為下列試驗之用。本研究主要利用 半量 MS (Murashige & Skoog 1962) 無機鹽類及其全量有機成分為基本配方 (1/2MS)，分別添加不 同濃度植物生長調節劑，加入凝膠物質 (Difco Bacto-agar) 前先以 0.1~1 N NaOH 及 HCl 將 pH 值 調至 5.7±0.1，再以 121℃、15 lb in-2 (1.05kg cm-2) 進行高溫高壓滅菌 15 min 後冷卻備用。 IBA 對丹參組培芽體發根之影響 利用高約 1.5-2 cm 之丹參組培芽體於添加不同濃度 IBA (0、0.25、0.5 及 1 mg L-1) 之 1/2 MS 固態培養基，置於 25±1℃恆溫、照光 (約 38 umol m-2 s-1) 16 小時之環境，經培養 4 週後調查組培 苗發根率與根數。 培養容器透氣性封口對丹參組培芽體發根之影響 利用高約 1.5-2 cm 之丹參組培芽體於內含 25 mL 培養基之 125 mL 三角瓶（Pyrex，日本）中， 培養基為 1/2MS 基本配方添加 0.5 mg L-1 IBA，三角瓶分別以兩層鋁箔紙 (aluminum foil; AF) 或 3 層藥包紙（9.5 × 9.5 × 0.046 cm，氣體流速 0.5 mL s-1，正隆，臺灣）(dispense paper; DP) 作為封口 之用。對照組全程 6 週均以兩層鋁箔紙作為容器封口 (AF6)，處理組則是於鋁箔紙封口培養 2 週後， 以三層藥包紙置換，再經 4 週培養 (AF2+ DP4)。在接種培養 6 週後調查芽體數、組培苗發根率與 根數。 丹參組培苗之移植馴化處理 將上述試驗已發根之丹參組培苗（高約 5-6 cm）出瓶，利用自來水洗淨根部附著之培養基，並 用稀釋 1000 倍免賴得 (Benomyl, 50% WP, Du Pont Co.) 消毒浸泡 30 分鐘後，除去部分葉片後移植 於直徑 9 cm 黑色塑膠盆，盆內為已滅菌且充分濕潤之 BioMix 培養土：蛭石：珍珠石 = 1：1：1 (v/v) 混 合介質。將種有組培苗之塑膠盆以透明塑膠袋覆蓋保持濕度，置入日、夜溫為 22℃，10h / 18℃， 14 h，與光強度約 60 umol m-2s-1 之生長箱進行馴化，四星期後移出至室溫下培養並調查存活率。每 處理 4 重複，每重複移植 6 株組培苗。成活植株於室外環境盆栽 3 個月後移至農試所田間栽培，丹 參植株於田間生長達 6 個月後，收穫地上部與根部，供做後續丹參酮含量分析之用。.
(3) 丹參組培苗馴化與丹參酮含量. 23. 丹參酮之含量測定 丹參酮類 (tanshinones) 含量測定係利用高效液相層析 (HPLC) 法，採用沃特斯公司相關儀器 設備（Waters，美國） ；層析管柱 A：RP-18, Inertsil ODS-3 (5 μm，4.6 × 33 mm)（GL Sciences Inc.， 日本） ；層析管柱 B：RP-18, Inertsil ODS-3 (5 μm，4.6 × 250 mm)（GL Sciences Inc.，日本）。分析 方法修改自 Chen et al. (1997)，在 30℃控溫條件下，以 Methanol：Tetrahydrofuran：Glacial Acetic Acid：H2O = 21.5：37.5：1：40 (v/v) 為移動相 (mobile phase)，流速 1 mL min-1，檢測波長為 254 nm。標準品丹參酮 I (Tan-I)、丹參酮ⅡA (Tan-IIA) 與隱丹參酮 (Crypto) 購自九鼎生物科技公 司，標準溶液配製濃度為 100、80、60、40 及 20 mg L-1，並以 0.45 μm 濾膜過濾備用。在上述的 層析條件下每次注射 10 μl 進行測定，每個濃度重複 3 次，再以成分濃度為橫座標，鋒面積為縱座 標，求得線性迴歸方程式及決定係數 (coefficient of determination; R2)。丹參檢品包括市售藥材（購 自彰化縣員林鎮）與組培苗移植栽培於農業試驗所田間 6 個月之植株地上部與根部，其中根部並依 其外觀顏色分為紅根及白根分別檢測。丹參檢品先以 50℃ 烘箱烘乾 48 hr，乾燥檢品取出研碎後 以適量 methanol 為溶媒浸漬過夜，經超音波洗淨器震盪 20-30 min，過濾並收集濾液，殘渣再加溶 媒重複萃取三次。將濾液混合以真空減壓濃縮機濃縮至完全乾燥，而後再以甲醇 (methanol; MeOH) 將其成分溶出且定量，再以 0.45 μm 濾膜過濾以供 HPLC 分析之用。各種丹參檢品注射 10 μl 檢品 濾液、重複注射 3 次，依所得線性迴歸方程式推算檢品中 Tan-I、Tan-IIA 及 Crypto 的含量。 資料分析 本研究發根試驗之每處理為 40 個組培芽體（每處理含 4 個重複，每重複含 10 個組培芽體） 。 培養容器封口試驗之每處理為 60 個組培芽體（每處理含 4 個重複，每重複含 15 個組培芽體） 。馴 化試驗每處理均為 36 株組培組培苗（每處理含 4 個重複，每重複含 9 株組培苗） ；試驗所得資料經 SAS 8.2 (SAS Institute Inc. 2001) 套裝統計分析軟體進行 ANOVA 變方分析，若處理間差異顯著， 則利用 Least significant difference (LSD) test 比較各處理平均值間差異。. 結. 果. IBA 對丹參組培芽體發根之影響 利用丹參組培芽體進行發根試驗結果如表 1 所示，添加 IBA 顯著促進丹參組培芽體不定根發 生率及增加不定根數目。其中以添加 0.5 mgL-1 IBA 處理可達較高發根率 (75%) 及較多根數（4.8 條/組培苗） ，且其根系發育也較為粗壯（圖 1A） 。雖然 1 mgL-1 IBA 處理之根數亦達 4.5 條/組培苗， 但其發根率下降至 65%。因此後續均以添加 0.5 mgL-1 IBA 之 1/2MS 培養基進行組培芽體發根培養。 培養容器封口資材對丹參組培芽體生長、不定根形成及組培苗移植成活率之影響 利用藥包紙置換鋁箔紙作為培養容器封口試驗結果顯示，容器封口置換對組培苗數之增減並無 顯著差異（表 2），但可提高組培芽體發根率 (90%)，對照組 (AF6) 之發根率僅 76.7%；就根數而 言，AF2+DP4 處理之 4.4 條/組培苗亦較 AF6 處理之 3.1 條/培植體為高，兩種發根指標均達顯著差 異（表 2）。觀察植株外觀結果顯示，藉由透氣性封口處理 (AF2+DP4) 組培苗具有較大葉片且健 壯之生長勢（圖 1B）。.
(4) 24. 台灣農業研究. 第 56 卷. 第 1 期. 表 1. 吲哆丁酸 (IBA) 濃度對丹參組培芽體發根之影響 Table 1. Influence of indole-3-butyric acid (IBA) on root formation in in vitro shoot of Salvia miltiorrhiza z IBA (mgL-1). Rooting (%) x. Roots per shoot y. 0. 37.5± 6.3 d. 2.9 ± 0.36 c. 0.25. 52.5±11.1 c. 3.8 ± 0.48 b. 0.5. 75.0± 5.0 a. 4.8 ± 0.35 a. 1. 65.0± 2.9 b. 4.5 ± 0.24 a. z. Twenty shoots per treatment were tested and the data was collected after 4 wks of culture. Means ± SE within a column followed by the same letters are not significantly different at the 5% level by LSD test.. 表 2. 培養容器封口對丹參組培芽體發根與組培苗移植存活率之影響 Table 2. Influence of container closure on root formation in in vitro shoots and survival rate in plantlets of Salvia miltiorrhiza z Treatment y. Shoots production. Rooting (%) x. Roots per shoot x. Survival rate (%) w. AF6. 3.6 ± 0.4 a. 76.7 ± 5.77 b. 3.1 ± 0.25 b. 27.8 ± 7.2 b. AF2＋DP4. 3.5 ± 0.2 a. 90.0 ± 4.30 a. 4.4 ± 0.33 a. 80.1 ± 5.3 a. z. In vitro grown shoots (about 1.5 cm long) were cultured on half-strength MS medium containing 0.5 mgL-1 IBA and 3% sucrose for 6 wks. Means ± SE within a column followed by the same letters are not significantly different at the 5% level by LSD test. y AF6: using 2 layers of aluminum foil as container closure for 6 weeks; AF2+DP4: Using 2 layers of aluminum foil as container closure for 2 wks, replacing with 3 layers of dispense papers for another 4 wks. x Thirty-six shoots were tested per treatment and the data was collected after 4 wks of culture. Roots shorter than 0.5 cm in length were not scored. w. Survival rate was recorded 4 wks after transferring into potting mixed substance in growth chamber for acclimation.. 將上述處理所得丹參組培苗出瓶上盆後，移入生長箱培養 4 週後調查存活率，結果顯示 AF2+DP4 處理之組培苗具有較高移植存活率 (80.1%)，而對照組 (AF6) 僅為 27.8%（表 2），且 AF6 處理組培苗外觀上顯得較為瘦弱。圖 1C 與 1D 分別顯示丹參組培苗出瓶種植 4 週與 12 週後植 株之生長情形。而後將馴化成活且生長良好之丹參苗種植田間，植株於田間生長達 6 個月後，採收 作為後續丹參酮類含量分析之用（圖 1E-F）。 丹參檢品之丹參酮含量分析 利用丹參酮類 (Tan-I、Tan-IIA 與 Crypto) 標準品溶液，進行 HPLC 定性與定量分析，結果顯 示三種成分之檢量線性方程式決定係數 (R2) 均達 0.95 以上，三者滯留時間 (retention time；RT) 分 別約於 13.6、22.5 與 11.4 min (表 3)。在丹參各檢品中以組培苗於田間栽培後植株之紅根所含 Tan-I+Tan-IIA+Crypto 較高，可達 1.85 mg g-1 dw，是丹參市場品含量的 1.5 倍。此外，在白根中 Tan-I+Tan-IIA+Crypto 含量亦可達 0.92 mg g-1 dw。而組培苗於田間栽培後植株地上部僅測得 Tan-IIA，含量為 0.02 mg g-1 dw（表 4） 。.
(5) 丹參組培苗馴化與丹參酮含量. 25. 圖 1. 丹參組培芽體發根與組培苗移植馴化過程中之植株生長情形。丹參組培芽體於添加 0.5 mg L-l 與 1 mg L-1 IBA 之 1/2MS 基本培養基 4 週後之發根情形 (A)；組培芽體培養於添加 0.5 mgL-1 IBA 之 1/2MS 基本培養基， 配合培養容器封口置換處理 (AF2+DP4) 達 6 週後，生長良好之發根組培苗 (B)；移植馴化處理 4 週，Bar = 2 cm (C)與 12 週，Bar = 5 cm (D) 後之丹參植株生長情形；丹參植株於農業試驗所田間達 6 個月之生長情形，Bar = 10 cm (E)；與採收時植株之地上部與根部，Bar = 5 cm (F)。 Fig. 1. In vitro shoots rooting and ex vitro plantlets acclimation of Salvia miltiorrhiza plantlets in field. Rooting of in vitro shoots of Salvia miltiorrhiza after culture on half-strength MS medium containing 1 or 0.5 mg L-1 IBA for 6 wks (A); Plantlets, on half-strength MS medium containing 0.5 mg L-1 IBA, containers capped with 2 layers of aluminum foil for 2 wks (AF2) then replaced by 3 layers of dispense paper for 4 wks (DP4) (total 6 wks incubation) (B); then acclimatized for 4 wks (C), Bar = 2 cm and 12 wks (D), Bar = 5 cm. Micropropagated plants of Salvia miltiorrhiza growing at the Institute farm for six months (E), Bar = 10 cm; and the aerial and root parts of harvested plants (F), Bar = 5 cm..
(6) 26. 第 56 卷. 台灣農業研究. 第 1 期. 表 3. 丹參酮類標準品迴歸檢量方程式、決定係數與其滯留時間 Table 3. The linear equation, coefficient of determination (R2), and retention time of tanshinones analysis by RP-HPLC method z R2. Retention time (min). 7. 5. 0.949. 13.63 ± 0.009. Tanshinone IIA. 7. 4. y =3.73×10 x – 2.10×10. 0.995. 22.47 ± 0.022. Cryptotanshinone. y =3.17×107 x – 1.58×103. 0.996. 11.38 ± 0.006. Tanshinones. Liner equation. Tanshinone I. z. y =3.00×10 x + 1.99×10. Samples were separated by C18 column with methanol: tetrahydrofuran: glacial acetic acid: H2O = 21.5: 37.5: 1: 40 (v/v) as mobile phase.. 表 4、丹參組培苗田間栽培植株地上部與根部及丹參市場品之丹參酮類含量分析 Table 4. Tanshinones content of aerial parts and roots derived from field-grew in vitro micropropagated plant of Salvia miltiorrhiza and Radix Salvia (market product, roots of Salvia miltiorrhiza) z Samples. Tan I. Tanshinones content (mg g-1 dry wt) z Tan IIA Crypto. Sum. Field grown plant Aerial part. nd. 0.02 ± 0.002. nd. 0.02. White roots. 0.45 ± 0.025. 0.18 ± 0.012. 0.29 ± 0.115. 0.92. Red roots. 1.45 ± 0.020. 0.27 ± 0.001. 0.13 ± 0.021. 1.85. Radix Salvia. 0.39 ± 0.017. 0.44 ± 0.017. 0.37 ± 0.017. 1.20. z. Field-grew in vitro micropropagated plant of Salvia miltiorrhiza which had grown in pot for 3 months and following 6 months of culture in field. y Three injections (10 μl each) were tested for each sample. Means (Mean ± SE) within a column followed by the same letters were not significantly different at the 5% level by LSD test. nd = not detected.. 討. 論. 近年來，利用組織培養技術已成功大量繁殖許多重要藥用植物組培苗，例如人參、黃耆、柴胡、 當歸、紅豆杉、白芷、金線連等 (Nalawade et al. 2003; Nalawade & Tsay 2004; Sagare et al. 2000)。 Chen et al. (2005) 利用丹參莖節腋芽部位已建立丹參大量繁殖組培芽體，且利用改善封口透氣性解 決芽體玻璃質化問題，惟組培苗往往由於移植存活率低，造成實際應用的諸多限制。本研究藉由調 整培養基之 IBA 濃度以促進組培芽體發根，並在芽體發根階段利用透氣性封口降低瓶內相對溼度 以提高組培苗品質及移植存活率，成功地建立適合於丹參組培芽體發根與組培苗移植馴化之方法； 此外，利用 HPLC 分析組培苗田間栽植植株與市售丹參藥材之丹參酮含量，證明組培苗作為生產 丹參藥材之可行性。 組織培養繁殖研究常以 IAA、NAA 或 IBA 作為促進組培芽體發根之植物生長調節劑，其中又 以 IBA 與 NAA 之效果較佳 (Gaspar et al. 1996; George 1993; Moncousin 1991)。在甜菊 (Stevia.
(7) 丹參組培苗馴化與丹參酮含量. 27. rebaudiana) (Sivaram & Mukundan 2003) 組培芽體發根研究中顯示出，IBA 具有促進芽體發根的效 果，且濃度愈高效果愈佳。Sahoo & Chand (1998) 報導 1 mg L-1 IBA 對黃荊 (Vitex negundo) 組培 芽體發根效果較佳，較高 (2 mg L-1) 與較低 (0.5 mg L-1) 濃度均不利其發根。此外，高氏柴胡 (Bupleurum kaoi) 組培芽體發根研究也顯示，0.5 mg L-1 IBA 處理之組培芽體基部並無癒合組織形 成，且具有高發根率與根數 (Chen et al. 2004a；Chen et al. 2006b)。本研究結果顯示，0.5 mg L-1 IBA 處理對於丹參組培芽體發根之效果較佳 (表 1)，此結果與上述其他學者之研究結果相似，顯示適當 IBA 濃度有利於提高組培芽體的發根率與發根數。 組織培養大量繁殖技術已廣泛應用於種苗生產事業，但因部分玻璃質化組培苗馴化存活率的低 下往往造成嚴重的成本負擔，雖然藉由一些化學與物理方法可抑制玻璃質苗形成或增進組培苗品 質，但也可能導致組培苗繁殖倍率的降低 (Chen et al. 2004b; Pâques 1991; Tsay 1998)。其中利用高 透氣性材質做為培養容器封口，係改善瓶苗品質之簡單且有效的方法，並已成功地應用於抑制康乃 馨 (Dianthus caryophyllus) (Chen et al. 1998) 與玄參 (Scrophularia yoshimurae) (Lai et al. 2005) 組 培芽體玻璃質化現象。此外，高氏柴胡 (Chen et al. 2004a; Chen et al. 2006b) 與玄參 (Chen et al. 2006a) 組培芽體利用透氣性培養容器封口之研究亦有相似的結果，不僅不影響組培芽體繁殖倍率 且可提高其組培苗移植存活率；藉由掃描式電子顯微鏡 (scanning electron microscope；SEM) 觀察 組培芽體與組培苗葉片結果顯示，氣孔功能正常與否與表皮蠟質累積多寡是影響移植存活率高低的 主要因子之一。本研究結果同樣指出，丹參組培芽體經培養於含 0.5 mg L-1 IBA 之 1/2MS 培養基， 在芽體發根培養階段藉由藥包紙進行 4 週透氣處理後並不影響芽體繁殖倍率，處理後之組培苗生長 健壯且根系形成良好（圖 1B），具有較高之移植存活率（表 3）。 當組培苗於盆栽馴化 3 個月後，再將其移植田間栽培達 6 個月時，即可顯示丹參植株生長相當 茂盛（圖 1E） ，採收後可發現其植株呈現特有的紅色根部（圖 1F） ，最大根部直徑達約 3.5 cm；經 HPLC 分析結果顯示其紅根之總丹參酮含量高於丹參市售藥材（表 4）。綜合以上結果顯示，田間 栽培丹參組培苗可於較短栽培時間內達到相當含量丹參酮的藥材，若能配合優良農業操作生產體系 (good agricultural practice; GAP) 規範栽培生產，則可獲得優質的丹參原物料，有利於臺灣藥用植物 產業之發展。. 誌. 謝. 本試驗研究承農委會農業生物技術國家型計畫經費補助 (91 農科-3.1.3-農-C1、92 農科-4.2.3農-C1、93 農科-4.2.1-糧-Z1)，以及朝陽科技大學博士後研究 Dinesh Chandra Agrawal 博士於文稿英 文部分之潤飾，特此申謝。. 引用文獻 (Literature cited) Caldas, E. D., and L. L. Machado. 2004. Cadmium, mercury and lead in medicinal herbs in Brazil. Food Chem. Toxicol. 42:599-603..
(8) 28. 台灣農業研究. 第 56 卷. 第 1 期. Chen, H., J. P. Yuan, F. Chen, Y. L. Zhang, and J. Y. Song. 1997. Tanshinone production in Ti-transformed Salvia miltiorrhiza cell suspension cultures. J. Biotech. 58:147-156. Chen, U. C., C. N. Hsia, D. C. Agrawal, and H. S. Tsay. 2006a. Influence of ventilation closures on plant growth parameters, acclimation and anatomy of leaf surface in Scrophularia yoshimurae Yamazaki - a medicinal plant native to Taiwan. Bot. Studies 47:259-266. Chen, U. C., C. N. Hsia, M. S. Yeh, and H. S. Tsay. 2004a. Influence of salts strength, sucrose, auxins and container closure on root formation and acclimation of in vitro Bupleurum kaoi plantlets. J. Agric. Res. China 53:249-260. (in Chinese with English abstract) Chen, U. C., C. N. Hsia, M. S. Yeh, and H. S. Tsay. 2006b. In vitro micropropagation and ex vitro acclimation of Bupleurum kaoi- a native endangerous medicinal plant. In Vitro Cell. Dev. Biol. 42:128-133. Chen, U. C., Y. J. Shiau, C. C. Lai, and H. S. Tsay. 1998. Effects of medium composition and vessel closure on the hyperhydricity and rooting of carnation in vitro culture. J. Agric. Res. China 47:346-376. (in Chinese with English abstract) Chen, U. C., M. S. Yeh, and H. S. Tsay. 2004b. Studies on in vitro shoot multiplication of native medicinal herbs- Bupleurum kaoi Liu, Chao et Chuang by axillary bud culture. J. Agric. Res. China 53:27-38. (in Chinese with English abstract) Chen, U. C., Y. J. Shiau, H. S. Tsay, and C. N. Hsia. 2005. Influence of cytokinin and ventilating container closure on shoot proliferation and hyperhydricity of in vitro Salvia miltiorrhiza culture. J. Agric. Res. China 54:93-102. (in Chinese with English abstract) Gaspar, T., C. Kevers, C. Penel, H. Greppin, D. M. Reid, and T. A. Thorpe. 1996. Plant hormones and plant growth regulators in plant tissue culture. In Vitro Cell. Dev. Biol. 32(P): 272-289. George, E. F. 1993. Plant propagation by tissue culture, Part 1. The technology. England: Exegetics Ltd. 574 pp. Guo, B. L., Y. X. Feng, and Y. J. Zhao. 2002. Review of germplasm resources studies on Salvia miltiorriza. China J. Chinese Materia Medica 27:492-495. (in Chinese with English abstract) Hu, Z. B., D. Liu, and A. W. Alfermann. 1999. Genetic transformation of Salvia miltiorrhiza. p.249-260. in: Biotechnology in Agriculture and Forestry 45, Transgenetic Medical Plants (Bajaj, Y. P. S. ed.). Springer-Verlag, Berlin. Lai, C. C., H. M. Lin, S. M. Nalawade, W. Fang, and H. S. Tsay. 2005. Hyperhydricity in shoot culture of Scrophularia yoshimurae can be effectively reduced by ventilation of culture vessels. J. Plant Physiol. 162:355-361. Moncousin, Ch. 1991. Rooting of microcuttings: general aspects. Acta Hortic. 289:301-310. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15:473-497. Nalawade, S. M., A. P. Sagare, C. Y. Lee, C. L. Kao, and H. S. Tsay. 2003. Studies on tissue culture of Chinese medicinal plant resources in Taiwan and their sustainable utilization. Bot. Bull. Acad. Sin. 44:79-98..
(9) 丹參組培苗馴化與丹參酮含量. 29. Nalawade, S. M., and H. S. Tsay. 2004. In vitro propagation of some important Chinese medicinal plants and their sustainable usage. In Vitro Cell. Dev. Biol. 40(P):143-154. Pâques, M. 1991. Vitrification and micropropagation: causes, remedies and prospects. Acta Hortic. 289: 283-290. Park, S., J. S. Song, D. K. Lee, and C. H. Yang. 1999. Suppression of AP-1 activity by tanshinone and cancer cell growth inhibition. Bull. Korean Chem. Soc. 20: 925-928. Sagare, A. P., Y. L. Lee, and H. S. Tsay. 2000. Application of biotechnological tools in conservation and utilization of medical plant genetic resource. p.171-187. in: Proceedings Agriculture of the New Century: Managing Bio-research and Bio-diversity. (Wu, W. S., S. T. Chang, and B. T. Guan eds.) College of Agriculture, National Taiwan University, Taipei, Taiwan. Sahoo, Y. and P. K. Chand. 1998. Micropropagation of Vitex negundo L., a woody aromatic medicinal shrub, through high-frequence axillary shoot proliferation. Plant Cell Rep. 18:301-307. Saper, R. B., S. N. Kales, J. Paquin, M. J. Burns, D. M. Eisenberg, R. B. Davis, and R. S. Phillips. 2004. Heavy Metal Content of Ayurvedic Herbal Medicine Products. Journal American Medical Association (JAMA) 292:2868-2873. SAS Institute Inc. 2001. SAS/STAT User’s Guide. Version 8.2, vol 2. SAS Inst., Cary, NC, USA. 943 pp. Sivaram, L., and U. Mukundan. 2003. In vitro culture studies on Stevia rebaudiana. In Vitro Cell. Dev. Biol. 39(P):520-523. Song, J. Y., Y. J. Zhang, and J. J. Qi. 1998. Biotechnology of Salvia miltiorrhiza. Natural Product Reseearch development 11:86-89. (in Chinese with English abstract) Tsay, H. S. 1998. Effects of medium composition at different precultures on vitrification of carnation (Dianthus caryophyllus) in in vitro shoot proliferation. Acta Hortic. 461: 243-249. Wu, Y. J., C. Y. Hong, S. J. Lin, and M. S. Shiao. 1998. Increase of vitamin E content in LDL and reduction of atherosclerosis in cholesterol-fed rabbits by a water-soluble antioxidant-rich fraction of Salvia miltiorrhiza. Arterioscler. Thromb. Vasc. Biol. 18:481-486..
(10) 30. 台灣農業研究. 第 56 卷. 第 1 期. In Vitro Plantlets Acclimation and Tanshinones Analysis of Salvia Miltiorrhiza1 Uei-Chern Chen 2, Chin-Yi Tsao 2 and Chi-Ni Hsia 2, 3 Abstract Chen, U. C., C. Y. Tsao, and C. N. Hsia. 2007. In vitro plantlets acclimation and tanshinones analysis of Salvia miltiorrhiza. J.Taiwan Agric. Res. 56:21-30. An efficient micropropagation protocol for producing high quality plantlets that are easy to acclimatize in field was developed for Salvia miltiorrhiza. In vitro grown shoots induced rooting (90%) after culturing on half-strength Murashige and Skoog’s basal medium (1/2MS) supplemented with 0.5 mg L-1 indole-3-butyric acid (IBA). Culture vessels were capped with aluminum foil for 2 weeks, and thereafter replaced with 3 layers of dispense paper for 4 weeks (AF2+DP4) were better than controls capped with aluminum foil for 6 weeks (AF6). Rooted shoots were transplanted into plastic pot containing a mixture of BioMix: vermiculite: perlite (1: 1: 1 ratio) (v/v). For acclimation, these pots were covered with transparent plastic bags and kept in a growth chamber under 14 h light at 22℃ and 10 h dark at 18℃ for 4 weeks. At 4 weeks of observation, these conditions resulted in the highest survival rate of 80.1% compared to AF6 treatment (27.8%). To compare, tanshinones contents (tanshinone-I, tanshinone-IIA and cryptotanshinone), the bioactive compounds in Salvia miltiorrhiza were estimated in the aerial parts and roots between in vitro micropropagated plants and market crude drug of S. miltiorrhiza by high performance liquid chromatography (HPLC). The quantity of tanshinones in red roots of in vitro micropropagated plants was measured as 1.85 mg g-1 dw about 1.5-fold higher than market crude drug. Key Words: Danshen, Medicinal plant, Micropropagation, High performance liquid hromatography (HPLC), Secondary metabolite.. 1. Contribution No. 2277 from Agricultural Research Institute (ARI), Council of Agriculture. Accepted January 25, 2007. 2. Assistant Agronomist, Contract Employees and Associate Researcher, Agronomy Division, ARI, Wufeng, Taichung, Taiwan, ROC. 3. Corresponding author, e-mail: hsia@wufeng.tari.gov.tw; Fax: (04)23302806..
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