丹參多倍體毛狀根誘導研究

丹參（Salvia miltiorrhiza）為唇形科多年生草本植物，其根部含有脂溶性之丹參酮，是中醫治療心血管疾病的重要藥材。多倍體毛狀根具有快速生長以及二次代謝物含量較高之特性，具有大量生產植物二次代謝物之潛力。本研究的目的在建立一個高效率的丹參多倍體毛狀根誘導方法，進而從中篩選出高丹參酮產量之多倍體毛狀根系，作為離體培養大量生產丹參藥用成分之用。本研究以二倍體之丹參轉殖毛狀根為材料，培養於含有2.5mM秋水仙素之1/2MS鹽類固態培養基中，進行2、4及6d不同天數的培養，結果顯示毛狀根之新根再生率以秋水仙素處理2d者最高，達80％；取再生新根以流式細胞儀進行染色體倍數檢測，並以2N丹參植株之葉片作為參考值，標示為2C（complement quantity），結果顯示以2d及4d處理得到之4C毛狀根誘導率較高，在52.4－55％之間。此外，將毛狀根區分為根尖與根段兩種培植體，培養於含有1.25mM及2.5mM秋水仙素之液體培養基中，分別震盪培養1、2及3d，結果顯示根尖培植體之新根再生率及4C毛狀根誘導率皆顯著高於根段培植體；秋水仙素1.25mM及2.5mM兩種濃度對毛狀根再生率之影響差異不顯著；但秋水仙素處理天數對新根再生率、4C毛狀根誘導率皆有顯著影響，新根再生率以2d處理最佳，4C毛狀根誘導率則以2d或3d處理最佳。綜合以上之結果，顯示以二倍體丹參毛狀根之根尖作為多倍體誘導材料，在含有2.5mM秋水仙素之固態或1.25mM秋水仙素之液態培養基中培養2d，可得到平均高於55％之4C毛狀根誘導率。進一步將為數33個之4C毛狀根系進行8wk培養，初步結果顯示，4C毛狀根系之平均生質量僅及2C毛狀根之0.49倍，但4C丹參毛狀根之隱丹參酮、丹參酮Ⅰ與丹參酮ⅡA的平均含量均較2C毛狀根為高，隱丹參酮增加最高達11.2倍，丹參酮之總產量則提高至2.6倍。

關鍵字

丹參；毛狀根；秋水仙素；多倍體；丹參酮

並列摘要

Salvia miltiorrhiza Bunge is a perennial herb of Labiatae Family. Its roots contain rich tanshinones and have been used for long history in traditional Chinese medicine to cure cardiology disease. Using fast growth hairy roots is becoming a new strategy for production of valuable secondary metabolites. Application of polyploidy hairy roots may provide an advanced approach to enhance in vitro secondary metabolite production. The objective of this study was to establish an efficient polyploidy induction method for S. miltiorrhiza to generate sufficient amounts of polyploidy hairy roots for selection of high medicinal content hairy root lines. Hairy roots induced from in vitro leaves of dipoloid S. miltiorrhiza plants were used as induction explants. Ploidy of regenerated roots derived from colchicine treatments were analyzed using a flow cytometry. A leaf from a 2 N plant was used as the control for flow cytometry which would indicate as 2 C (complement quantity). Root explants were cultured in a solid medium containing with 2.5 mM colchicine for 2, 4 and 6 days to induce polyploidy hairy roots. Treatment of 2-day-exposure colchicines had the highest root formation rate of 80%. The highest polyploidy induction rate of 52.4-55% was obtained from 2- and 4-day-exposure of colchicine. Furthermore, root explants of tips and sections were used as induction explants incubated in a liquid medium containing 1.25 mM and 2.5 mM colchicine for 1, 2 and 3 days to induce polyploidy. It was found that explant type and exposure duration of colchicine had significant effects on root formation and polyploidy induction rate however effect of colchicine concentration was found not significantly. The highest root formation rate was found from root tip explant for 2-day-exposure. The highest 4 C induction rates were found from either 2- or 3-day-exposure of the tip explant. In conclusion, root tips are more suitable explants than that of root sections for in vitro polyploidy induction. Root tip explant incubated in a solid medium containing 2.5 mM colchicines or in a liquid medium containing 1.25 mM colchicines for 2-day-exposure had the 4C hairy root induction rate higher than 55%. A total 33 lines of 4C hairy roots were subcultured to a new medium for 8 weeks of culture. The average biomass obtained from 4C hairy root lines showed only in 49% of the diploid hairy roots. In contrast, the average contents of cryptotanshinone, tanshinone I, and tanshinone IIA obtained from 4 C hairy root lines were all found higher than that of the diploid hairy roots. Moreover, an 11.2 folds increasing of the cryptotanshinone contents obtained from 4 C hairy root lines and making a 2.6 folds increasing for the total tanshinone production than that of the 2 C hairy roots.

並列關鍵字

Salvia miltiorrhiza Bunge ； Hairy root ； Colchicine ； Polyploidy ； Tanshinone

國際替代計量

丹參多倍體毛狀根誘導研究

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