生長素與通氣處理對仙草組織培養苗增殖與生育之影響

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(1)台灣農業研究 (J. Taiwan Agric. Res.) 60(4):293–299 (2011). 仙草組織培養菌之大量繁殖. 293. 生長素與通氣處理對仙草組織培養苗增殖與生育之影響. 1. 羅淑卿2 蔡新聲3,4 摘. 要. 羅淑卿、蔡新聲。2011。生長素與通氣處理對仙草組織培養苗增殖與生育之影響。台灣農業研究 60:293–299。. 本研究探討不同生長激素 [NAA (α-naphthaleneacetic acid)、IAA (indole-3-ace tic acid) 及 IBA (indole-3-butyric acid)] 及透氣處理對仙草組培苗育成之影響。莖頂培植體接種於 0.5 mg/L NAA 之 1/2 MS 固體培養基，暗培養 4 週所誘導的團粒狀癒合組織，再經光照 4 週培養後可形成叢生芽，以供仙草種苗大量繁殖利用；將叢生芽截切為單節培植體，移植至含 0.5 mg/L 之 NAA、IBA 或 IAA 之 1/2 MS 基礎鹽類培養基，於 25℃、光照 30–35 μmol m-2 s-1、培養 6 週以比較植株生育情形；於另一個試驗探討不同瓶口覆蓋物及透氣時間處理對培養於含 0.5 mg/L IBA 之 1/2 MS 基礎鹽類培養基之植株苗玻璃質化的影響，結果顯示 3 個生長素處理，以 IBA 可得生長較佳的瓶苗，根重為 0.031 克/株，而 IAA 處理者只有 0.011 克/株；透氣試驗以 4 層藥包紙覆蓋瓶口，再覆以石蠟膜以減少透氣，經培養 5 週後再除去石蠟膜以透氣者，可得移植存活率高之優質組培苗，移植存活率為 98.7%，較鋁箔處理者 92.1%為高。藉由生長素 IBA 的添加及透氣處理可育成健康、生長良好之仙草組培苗，此技術可應用於增殖種苗，並免除非季節性佔用種植土地之情形。關鍵詞︰仙草、組織培養、生長素、透氣處理。. 前. 言. 仙草 (Mesona procumbens Hemsl.) 屬唇形科高經濟價值之特用作物，依據古代文獻中藥大辭典記載，仙草味澀、甘、寒，具有清熱、解渴、涼血及降血壓之功效，可治臟腑熱毒 (Hung 2001)，說明仙草是具生理機能性之中草藥。仙草的許多療效目前均已得到科學證實； Capellades (1990) 報告指出，仙草水萃取物具 1. 2. 3. 4.. 有良好的清除自由基及抗氧化能力，內含熊果酸 (ursolic acid)、齊墩果酸 (oleanolic acid) 等三萜類 (triterpenes) 成分，這些成分在文獻上均指出具減緩急慢性肝炎、抗發炎、抗高血脂及抗腫瘤之生理功效 (Liu 1995)。仙草栽培之種苗來源慣以扦插育苗以防種質變異，在非種植季節仍需佔地種植母株，若以育成組織培養瓶苗的技術增殖種苗，則可省去種植母株土地空間，並可避免天候病蟲害等不確定之傷害種. 行政院農業委員會農業試驗所研究報告第 2603 號。接受日期：100 年 8 月 31 日。本所農業化學組助理研究員。台灣台中市。朝陽科技大學生化科技研究所講座教授。台灣台中市。通訊作者，電子郵件：hstsay@cyut.edu.tw；傳真機：(04) 23304921、23742371。.

(2) 294. 台灣農業研究. 苗因素。因此本研究擬利用農試所栽種之農試 1 號仙草品種，進行組織培養苗之繁殖試驗，探討苗體繁殖之合適條件，以供相關產業參考利用。. 材料與方法供試材料本試驗使用之材料，係採自農試所內栽種的農試 1 號仙草。. 癒合組織與不定芽誘導之觀察切取仙草莖頂組織長約 3 mm，以自來水沖洗乾淨後，於 70%酒精中浸泡 30 秒，再以 0.5% 次氯酸鈉 (每 100 mL 含 2–3 滴 Tween 20) 震盪消毒 10 分鐘，最後以無菌去離子水清洗 3–4 次，於無菌接種箱中切去白化的組織後，接種於含 0.5 mg/L NAA、3%蔗糖、0.9%洋菜 (Difco agar) 及 pH = 5.7 ± 0.1 之 1/2 MS (Murashige & Skoog 1962) 斜面固體培養基 (Cassells et al. 1994)，培養容器為 45-mL 試管，培養環境為 (25 ± 1)℃，進行暗培養以誘導癒合組織。莖頂組織經 4 週暗培養後形成團粒結構之癒合組織，移至每日照光 16 小時、光強度約 30–35 μmol m-2 s-1 之環境，約 1 個月後可誘導為不定芽。. 不同生長素對植株生長與移植存活試驗前項試驗誘導形成之不定芽切成含一分枝之節段作為培植體，分別接種於含 0.5 mg/L 之 NAA、IBA 或 IAA 之 3 種不同生長素 (auxin) 的 1/2 MS 培養基 (成分如前述)，培養容器為 125-mL 三角瓶，每種培養基各 10 瓶，瓶內各 5 個節段培植體，於 6 週後調查其育成瓶苗之農藝性狀，調查項目為株高、葉數、芽體數、根重、植株鮮重。將上述不同培養基之組培瓶苗，分別移出栽種於經高溫高壓滅菌之泥炭土、蛭石 (3 號)、珍珠石 (4 號) 1：1：1 (v/v) 之混合介質，不同處理均各種植 3 盤，於 1 個月後調查其存活率。. 第 60 卷. 第4期. 不同透氣處理對組培苗生長與移植存活試驗將與前項試驗相同之節段培植體接種於含 0.5 mg/L IBA 之 1/2 MS 固體培養基，瓶內各 5 個節段培植體，瓶口覆蓋 4 層滅過菌藥包紙，最後再以石蠟膜 (Parafilm M®) 包覆於藥包紙外，分別於培養 3、4、5、6 週後去除外部石蠟膜以續行透氣培養，達培養週期共 8 週 (代碼分別為 P3 + DP5、P4 + DP4、P5 + DP3 及 P6 + DP2)，取出調查組培苗之農藝性狀；對照組為以二層鋁箔封住瓶口，試驗全程期間皆不除去石蠟膜 (代碼 AF8)，每種透氣處理各 10 瓶 (Chen et al. 2006a; Tsay et al. 2006)。將上述不同透氣處理之組培瓶苗，分別移出栽種於經高溫高壓滅菌之泥炭土、蛭石 (3 號)、珍珠石 (4 號) 1：1：1 (v/v) 混合介質，不同透氣處理處理均各種植 3 盤，於 1 個月後調查其存活率。. 統計分析上述試驗資料均經 SAS 統計分析套裝軟體進行變方分析 (analysis of variance, ANOVA) 後，以最小顯著差異性測驗 (least significant difference test, LSD test)，在 5%顯著水準下比較各處理平均值間之差異，若為百分率之數據，則先經平方根轉換。. 結. 果. 不同生長素對植株生長與移植存活率之影響仙草莖頂接種於 1/2 MS 含 0.5 mg/L NAA 之培養基，基部會形成團粒結構之癒合組織，將此癒合組織移至照光環境下再經 4 週培養後，可誘導大量不定芽產生，平均每個莖頂約可誘導 10–20 個不定芽 (圖 1)，將此不定芽截切為多個含一個節之培植體，接種於含有不同生長素之培養基，結果顯示 3 種生長素對植株葉數、根重及鮮重等性狀，以 0.5 mg/L IBA 處理，效果顯著優於含 0.5 mg/L NAA 或 IAA 之.

(3) 仙草組織培養菌之大量繁殖. 295. 處理；移植出瓶後植株之存活率也以 0.5 mg/L IBA 為佳，達到 92.3%，由結果顯示，誘導仙草節段發根及生長之最佳培養基為 0.5 mg/L 之 IBA (表 1)。瓶苗植株外觀上，玻璃質化之情形極為普遍。. 不同透氣處理對組培苗生長之影響. 圖 1. 仙草癒合組織接種於 0.5 mg/L NAA 之 1/2 MS 固體培養基移至照光環境 4 週後，誘導出之不定芽生長情形。 Fig. 1. Development of Mesona procumbens plantlets induced by calluses placed under light for 4 weeks cultivated in half strength MS basal solid medium containing 0.5 mg/L NAA.. 為培育無玻璃質化之優質瓶苗，以上述試驗 0.5 mg/L IBA 處理之培養條件，進行透氣無菌培養，即捨棄鋁箔改以滅菌過的四層藥包紙封住瓶口外面再封以石臘膜，並分別在培養第 3、4、5、6 週後去除石蠟膜，以加大瓶內氣體和外界之通透率，經 8 週週期後，取出瓶苗作農藝性狀調查同時進行移植存活試驗，結果顯示以全程以鋁箔紙封瓶口之對照組，瓶苗有玻璃質化之現象，而藥包紙處理則只有封口 6 週者出現玻璃質化，且不論莖長、葉寬、根重及植株鮮重皆以在第 5 週去除石蠟膜者為佳，且較其它組有顯著性差異，取出之瓶苗進行移植試驗時，亦以第 5 週者存活率最高，較其它組有顯著性差異 (表 2)；其次以第 4 週處理根重大於第 6 週，其存活率也較優於第 6 週 (表 2)。本試驗結果亦顯示，太晚進行通氣處理不利於仙草瓶苗之生長，且仍出現玻璃質化，但太早通氣 (第 3 週) 則易使培養基失水龜裂，也不利於仙草瓶苗之生長 (圖 2)。. 表 1. 仙草不定芽以不同生長調節劑處理六週後之組培苗農藝性狀調查 Table 1. Agronomic characteristics of regenerated plantlets of Mesona procumbens grown on 1/2 MS medium amended with different auxins for 6 weeks z Auxins treatment. plantlet height. No. of leaves/. No. of shoots/. Root weight/. Fresh weight/. Survival rate. (cm). plantlet. plantlet. plantlet (g). plantlet (g). (%). IAA. 3.5 ± 0.3 b. IBA NAA z y. y. 17.4 ± 2.1 b. 2.4 ± 0.5 a. 0.011 ± 0.001 c. 0.42 ± 0.01 b. 87.5 ± 1.1 b. 4.3 ± 0.3 ab. 25.5 ± 2.9 a. 2.1 ± 0.3 ab. 0.031 ± 0.002 a. 0.62 ± 0.01 a. 92.3 ± 0.6 a. 4.9 ± 0.2 a. 17.5 ± 1.6 b. 1.1 ± 0.4 b. 0.021 ± 0.001 b. 0.29 ± 0.01 c. 91.3 ± 0.6 ab. Single nodes explants were cultured on half strength MS basal solid medium containing 0.5 mg/L of IAA, IBA or NAA. Values are mean ± SE (n = 10). Means with different letter(s) in the same column are significantly different (P < 0.05) by LSD test. Percentage data of survival rate were square-root transformed prior to analysis..

(4) 296. 台灣農業研究. 第 60 卷. 第4期. 表 2. 仙草苗培養行不同時間之透氣處理後之農藝性狀調查 Table 2. Agronomic characteristics of regenerated plantlets of Mesona procumbens grown under different gas exchange treatments Ventilation. Stem length. Leaf width. Root weight/plantlet. Fresh weight/Plantlet. closures z. (cm). (cm). (g). (g). Survival rate (%). 1.36 ± 0.09 b y. 1.04 ± 0.07 b. 0.021 ± 0.001 d. 0.500 ± 0.010 c. 92.1 ± 0.33 d. P3 + DP5. 1.32 ± 0.06 b. 1.05 ± 0.04 b. 0.030 ± 0.001 c. 0.504 ± 0.009 c. 92.9 ± 0.18 d. P4 + DP4. 1.44 ± 0.05 ab. 1.08 ± 0.04 ab. 0.041 ± 0.013 b. 0.602 ± 0.019 b. 97.2 ± 0.28 b. P5 + DP3. 1.59 ± 0.08 a. 1.21 ± 0.06 a. 0.051 ± 0.001 a. 0.718 ± 0.023 a. 98.7 ± 0.29 a. P6 + DP2. 1.35 ± 0.08 b. 1.06 ± 0.05 ab. 0.031 ± 0.001 c. 0.610 ± 0.021 b. 96.0 ± 0.24 c. AF8. z. y. AF8: The opening of each container was covered with two layers of aluminum foil (AF) during 8 weeks of incubation; P3 + DP5, P4 + DP4, P5 + DP3, and P6 + DP2: Two additional layers of Parafilm (P) were removed after 3, 4, 5, and 6 weeks of incubation, followed by four layers of dispense paper for the next 5, 4, 3, and 2 weeks of incubation. Values are mean ± SE (n = 10). Means with different letter(s) in the same column are significantly different (P < 0.05) by LSD test. Percentage data of survival rate were square-root transformed prior to analysis.. 圖 2. 仙草苗在 0.5 mg/L IBA 培養下行不同時間之透氣處理後之生長情形。 Fig. 2. Effect of duration of ventilation treatment on growth of Mesona procumbens seedlings cultivated on half strength MS basal solid medium containing 0.5 mg/L IBA. Note vigorous growth of seedlings in the treatments of 3-, 4- or 5-weeks old seedlings but poor growth in the treatment of 6-weeks old seedlings.. 討. 論. 利用組織培養大量繁殖種苗是商業種苗生產之重要技術，本試驗進行仙草瓶苗生長與發根試驗時，以 1/2 MS 基礎培養基分別添加 0.5 mg/L IAA、NAA、IBA 三種處理，皆可育成仙草瓶苗，存活率也都有 80%以上，觀察其仙草株型，在培養至第 3 週時，分枝大致已成型。量產組織培養種苗常利用 IAA、NAA、IBA 為促進組培苗發根之生長調節劑，其中又以 NAA 、 IBA 效果為佳 (Gaspar et al. 1996; Moncousin 1991)。由本研究可見以 IBA 處理. 者，根重大於 NAA 處理。以 IBA 處理者植株較重、株形發展均衡且根系形成較佳；亦有研究指出若 IBA 或 NAA 濃度太高，易造成基部癒合組織化現象 (basal callusing) (Chang et al. 2000)，會不利於後續的移植馴化成苗，在本研究中並未發生此現象，可見 0.5 g/mL IBA 為培育仙草瓶苗之適當生長調節劑及濃度；由於此試驗皆以鋁箔封住瓶口，不論何種處理皆出現植體玻璃質化 (hyperhydricity) 之情形，以鋁箔封住瓶口是為防止污染及培養基水分散失，但由於其材質透氣性低，導致培養瓶內濕度高、有害氣體 (如二氧化碳及乙烯) 及致毒形態氧.

(5) 仙草組織培養菌之大量繁殖. (toxic oxygen forms) 的累積，致使植體葉面氣孔及表皮臘質層的發育異常或喪失功能，如此造成苗體外觀似玻璃，透明易碎，移植馴化存活率不高 (Chen et al. 2006b)。為改善仙草組培苗玻璃質化現象，本試驗續探討通氣處理對瓶苗之育成影響，以上述生長較佳之 IBA 培養基條件接續以下試驗，摒除上述之鋁箔改用透氣性較高之 4 層藥包紙封口，藥包紙外部再加封 2–3 層石蠟膜，並分別在培養 3、4、5、6 週後，去除封在藥包紙上之石蠟膜，使氣體能通透，在第 8 週時取出瓶苗調查苗體之農藝性狀，顯現於第 3 週就去除石蠟膜者，由於過早透氣處理，易使培養基的水分散失，因而苗體在瓶內培植生長不良，莖短葉形小，移植存活率較差；第 5 週去除石蠟膜之瓶苗生長最健康，葉片較大，根系及植株發育佳，移植存活率亦提高；第 6 週才去除石蠟膜者，可見瓶苗則已出現玻璃質化之情形，本試驗結果與 Chen (1998) 康乃馨組培試驗有相同之情形；植體雖出現玻璃質化，但只要即時將氣體通透至瓶中，亦可以使植株回復為正常苗株，此玻璃質化苗移植於混合蛭石、泥炭土、珍珠石亦可回復為正常苗，存活率亦有 90%以上，顯示仙草生命力極為強健。影響玻璃質化的因素很多 (Chen et al. 1998)，玻璃質化之組培苗直接移植至溫室環境下存活率會下降，瓶內馴化 (acclimatized in vitro) 主要為降低瓶內相對溼度及增加氣體的交換率，如此可增加葉面臘質之形成及氣孔之功能維持，有助培養出健康苗體，於移植出瓶時易存活，因此多數研究報告利用通氣性佳的覆蓋材質，皆能有效控制植體玻璃質化情形發生及增加瓶苗移出馴化的存活率 (Capellades et al. 1990; Cassells & Wash 1994; Lai et al. 2005; Ritchie et al. 1991; Smith et al. 1990; Wardle et al. 1983; Yue ＆ Gosselins 1993; Ziv 1986; Ziv 1991)。依本研究結果顯示，1/2 MS. 297. 固體培養基、添加 0.5 mg/L IBA 並配合藥包紙封口之透氣處理，可有效防止仙草瓶苗玻璃質化之情形，並育成高存活率之優質仙草植株。. 引用文獻 (Literature cited) Capellades, M., R. Fontarnau, C. Carulla, and P. Debergh. 1990. Envionment influences anatomy of stomata and epidermal cells in tissue - cultured Rosa multiflora. J. Am. Soc. Hortic. Sci. 115:141–145. Cassells, A. C. and C. Wash. 1994. The influence of gas permeability of the culture lid on calcium uptake and stomatal function in Dianthus microplants. Plant Cell Tiss. Org. Cult. 37:171–178. Chang, S. H., C. K. Ho, J. Y. Tsai, and Z. Z. Chen. 2000. Thidiazuron enhancement of plant regeneration from leaf calli of superior clones of Eucalyptus camaldulensis. Taiwan J. For. Sci. 15:81–90。 Chen, U. C., C. N. Hsia, M. S. Yeh, 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 Stud. 47:259–266. Chen, U. C., C. N. Hsia, M. S. Yeh, D. C. Agrawal, and H. S. Tsay. 2006b. In vitro micropropagation and ex vitro acclimatization of Bupleurum kaoi - an endangered medicinal plant native to Taiwan. In Vitro Cell Dev. Biol. Plant. 42:128–133. Chen, U. C., Y. J. Shiar, C. C. Lai, and Tsay. H. S. 1998. Effect of medium composition and vessel closure on the hyperhydricity and rooting of carnation in vitro culture. J . A g r i c . Re s . C h i n a . 47:364– 376. (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. 32:272–289. Hung, C. Y. 2001. Studies on Antioxidant Functionality of Hsian-tsao (Mesona procumbens Hemsl.). Ph. D. Thesis. Graduate Institute of Food Science, National Chung Hsing Univ. Taichung. Taiwan. 203 pp. (in Chinese) 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..

(6) 298. 台灣農業研究. 第 60 卷. 第4期. Liu, J. 1995. Pharmacology of oleanolic acid and ursolic acid. J. Ethnopharmacol. 49:57–68.. medicinal plant. In Vitro Cell Dev. Biol. Plant. 42:445–449.. Moncousin, C. 1991. Rooting of microcuttings: general aspects. Acta Hortic. 289:301–310.. Wardle, K., E. B. Dobbs, and K. C. Short.1983. In vitro acclimatization of aseptically cultured plantlets to humidity. J. Am. Soc. Hortic. Sci. 108:386–389.. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15 :437–497. Ritchie, G. A., K. C. Short, and M. R. Davey. 1991. In vitro acclimatization of chrysanthemum and sugar beet plantlets by treatment with paclobutrazol and exposure to reduced humidity. J. Exp. 42:1557– 1563. Smith, E. F., A. V. Roberts, and J. Mottly. 1990. The preparation in vitro of chrysanthemum for transplantation to soil. 3. Improved resistance to desiccation conferred by reduced humidity. Plant Cell Tiss. Org. Cult. 21:141–145. Tsay, H. S., C. Y. Lee., D. C. Agrawal, and S. G. Basker. 2006. Influence of ventilation closure, gelling agent and explant type on shoot bud proliferation and hyperhydricity in Scrophularia yoshimurae - a. Yue, D. and A. Gosselins. 1993. Effects of forced ventilation at different relative humidity on growth, photosynthesis and transpiration of geranium plantlets in vitro. Can. J. Plant Sci. 73:249–256. Ziv, M. 1986. In vitro hardening and acclimatization of tissue culture plant. p.187–196. in: Plant Tissue Culture and its Agriculture Application. (Withers, L. A. and P. G. Alderson, eds.) Butterworths London. Ziv,. M. 1991. Vitrification: Morphological and physiological disorders of in vitro plant. p.45–69. in: Micropropagation: Techology and Application. (Debergh, P. C. and R. H. Zimmerman, eds.) Kluwer Academic Pub. Dorcreht..

(7) 仙草組織培養菌之大量繁殖. 299. Effects of Auxins and Ventilation on Growth and Multiplication of Plantlets of Mesona procumbens Hemsl.1 Shu-Chin Lo2 and Hsin-Sheng Tsay3,4 Abstract Lo, S. C. and H. S. Tsay. 2011. Effects of Auxins and Ventilation on Growth and Multiplication of Plantlets of Mesona procumbens Hemsl. J. Taiwan Agric. Res. 60:293–299.. This study was conducted to determine effects of auxins [NAA (α-naphthaleneacetic acid), IAA (indole-3-acetic acid) and IBA (indole-3-butyric acid)] and ventilation on in vitro micropropagation of Mesona procumbens Hemsl. Shoots of M. procumbens were grown in dark on 1/2 MS media supplemented with 0.5 mg/L of NAA for 4 weeks for formation of granule-like calluses and then placed under light for another 4 weeks for the development of plantlets. The plantlets were cut into segments, transferred on 1/2 MS media supplemented with 0.5 mg/L of NAA, IAA or IBA, and incubated at 25℃ under light 30–35 μmol m-2 s-1 for 6 weeks to compare effects of auxins on plantlet growth and development of plantlets. In another experiment, plantlets were transferred on 1/2 MS media supplemented with 0.5 mg/L of IBA to study effect of different container sealing materials and ventilation closures on hyperhydricity of plantlets. Results showed that among the three auxins tested, the medium containing 0.5 mg/L of IBA was most suitable for growth of plantlets of M. procumbens with a root weight of 0.031g/plant, compared to 0.011g/plant for the treatment of IAA. Ventilation affected growth of plantlets. When the tissue culture containers were sealed with dispense paper and Parafilm for 5 weeks and then removed the Parafilm for ventilation, the survival rate of ex vitro acclimation of plantlets increased to 98.7%, compared to 92.1% in the treatment of plantlets in aluminum foil. Thus, it is possible to produce healthy plantlets of M. procumbent in tissue culture with proper amount of IBA in the growth medium and proper control of ventilation. The protocol established in this study would be useful for in vitro propagation of M. procumbent and it could be a potential alternative to the conventional propagation of this crop in the field. Key words: Mesona procumbens, Tissue culture, Auxins, Ventilation.. 1. Contribution No. 2603 from Taiwan Agricultural Research Institute (TARI), Council of Agriculture. Accepted: August 31, 2011. 2. Assistance Researcher, Agricultural Chemistry Division, TARI, Taichung, Taiwan, ROC. 3. Professor, Chaoyang University of Technoloty, Taichung, Taiwan, ROC. 4. Corresponding author, e-mail:hstsay@cyut.edu.tw; Fax: (04)23304921, 23742371..

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