細本山葡萄毛狀根誘導、離體培養及白藜蘆醇生產

Induction of in Vitro Cultured Hairy Roots of Vitis Thunbergii Sieb. & Zucc. and Trans-Resveratrol Production

古國隆(Kuo-Lung Ku) ； 葉玟萱(Wen-Hsuan Yeh) ； 廖宇賡(Yue-Ken Liao)

《中華林學季刊》 46卷3期 (2013/09) Pp. 255-274

摘要

本研究建立細本山葡萄（Vitis thunbergii Sieb. & Zucc.）毛狀根誘導及培養方法，並初步測試誘引因子刺激生產反式白黎蘆醇（trans-resveratrol，t-Res）的效果。以不同農桿根群菌（Agrobacterium rhizogenes）菌系接種細本山葡萄無菌苗葉片（先端）後，不定根之發生以接種BCRC 15010者優於BCRC 15011。這些誘導而來的根群並非均由轉殖而產生，在6次的繼代過程中有90%以上遭淘汰。穩定存活者經polymerase chain reaction（PCR）增量特定基因片段確認為轉殖毛狀根後，依不同外觀形態選定Line 1-2（15010接種）及Line 4-1（15011接種）進行後續試驗。兩個line皆以1/4 mB5液體培養基添加2%蔗糖培養有最佳生長；且有利t-Res累積。兩個line於1盞uv燈（20 w）照射10 min後放置12 h可達t-Res累積高峰。在幾丁聚醣（chitosan）誘引試驗中，Line 1-2及4-1亦對濃度10 mg/L的處理產生反應，增加t-Res的含量。惟Line 4-1單獨對醋酸對照組（chitosan的溶劑）亦出現近似chitosan的誘引效果，其實際上受此誘引劑刺激後的反應並不顯著。

關鍵字

農桿根群菌 ； 誘引因子 ； 毛狀根 ； 反式白黎蘆醇 ； 細本山葡萄

並列摘要

The present study established an induction/culture system for hairy roots of Vitis thunbergii Sieb. & Zucc. and preliminary examined the effects of elicitor stimulation on trans-resveratrol (t-Res) production. Two strains of Agrobacterium rhizogenes (BCRC 15010 and 15011) were applied to inoculate the explants (distal-half leaves) of host plant and greater root induction was recorded in strain 15010. However, the newly induced adventitious roots were not all transformed by the agrobacteria. Over 90% of the root cultures ceased growing and were eliminated through prolonged subcultures. Two hairy-root cultures with different morphology, Lines 1-2 (15010 infected) and 4-1 (15011 infected), were selected based on identification of specific polymerase chain reaction (PCR) -amplified gene fragments and used as experimental materials. Both lines obtained greater performance in growth and I-Res production while incubating on 1/4 mB5 medium containing 2% sucrose. Ultraviolet (UV) light (20 W, 10 min illumination) was capable of increasing I-Res content to the maximum in both lines 12 h after exposure. The t-Res production was also enhanced by chitosan (10 mg/L) stimulation. However, Line 4-1 was less responsive to this elicitor due to its similar production of I-Res in acetic acid treatment (control), which was used as a solvent in this study for chitosan preparation.

並列關鍵字

Agrobacterium rhizogenes ； elicitor ； hairy root ； trans-resveratrol ； Vilis thunbergii