利用細本山葡萄細胞培養生產白黎蘆醇之研究

Production of resveratrol by cell culture of Vitis thunbergii Sieb.et Zucc.

郭賢伸(Hsien-shen Kuo)

指導教授 ： 何錦玟

大同大學/工程學院/生物工程學系所/碩士(2006年)

摘要

經三年長期繼代之細本山葡萄（Vitis thunbergii Sieb. et Zucc.）癒合組織（long-term callus），在不添加elicitor下進行懸浮培養18天後，細胞中resverarol含量有21.78 mg/kg DW，細胞鮮重增加約3.5倍。以生物性及非生物性因子刺激long -term callus累積resveratrol，在培養基中添加100 g/L Botrytis cinerea 滅過菌的菌絲，其細胞內有最高resveratrol含量（267.29±70.59 mg/kg DW，控制組為21.79±8.69 mg/kg DW），但細胞大量死亡；而在培養基中添加 6 mg/L methyl jasmonate可以讓細胞中resveratrol含量提升到198.59±66.95 mg/kg DW，並可細胞鮮重增加4倍（3g→12.57g）。照射強度30W的UV光30分鐘可以提升細胞中resverarol含量達110.18±34.72 mg/kg DW，對細胞生長沒有抑制。而提高培養基中蔗糖濃度到150 g/L也可以提升細胞中resveratrol含量（216.08±65.18 mg/kg DW），但卻不利細胞生長。
　　利用in vitro植株之不同培植體，培養於不同生長調節劑組合的MS培養基中誘導新癒合組織，結果以1.5 mg/L NAA 搭配 0.5 mg/L BA誘導效果最佳，在莖段及葉柄都可以明顯的誘導出癒合組織（new callus），新誘導癒合組織經三次繼代後測其resveratrol含量可達3616.36 mg/kg DW（葉柄）及 3208.37 mg/kg DW（莖段）。

　　長期繼代之細胞生長快速但resveratrol含量會明顯降低，利用elicitor處理雖可有效促進細胞內resveratrol增加2-10倍，但含量仍不及新誘導癒合組織的十分之一。顯示長期繼代細胞系其resveratrol生產能力會快速降低。

關鍵字

白黎蘆醇 ； 細本山葡萄 ； 組織培養

並列摘要

The callus of Vitis thunbergii Sieb. et Zucc. was subcultured bimonthly for three years (long-term cell) and was used to establish suspension culture and examine the content of resveratrol in cells. Biomass of long-term cell increased nearly 3.5 times every 18 days cultured in 1/2 MS medium supplemented with 1.86 mg/L NAA and 0.22 mg/L BA. Biotic factors (fungi hypha and yeast elicitor) and abiotic factors (jasmonic acid , methyl jasmonate , salicylic acid, phenylalanine, cinnamic acid, UV ) were used to treat the cells to enhance accumulation of resveratrol. The resveratrol content of cells reached to 267.29±70.59 mg/kg DW (the control was 21.79±8.69 mg/kg DW) when cell cultured in modified MS medium contained autoclaved hypha of Botyis cinerea, but the cells dead quickly. Methyl jasmonate at 6 mg/L in medium, resveratrol content boosted to 198.59±66.95 mg/kg DW and the fresh weight of biomass to 12.57 g (original inoculum was 3 g) for 18 days culture. Cells treated with UV (power:30W, time:30 minutes) proliferated normally and the resveratrol content in cells was 110.18±34.72 mg/ kg DW. Addition of high dose of sucrose (150 g/L) cause resveratrol accumulation (216.08±65.18 mg/kg DW) but cells eventually died.
The fast growing callus induced from stem and petiole were cultured on modified MS medium with 1.5 mg/L NAA+ 0.5 mg/L BA (new callus). After three subcultures, the content of resveratrol in new callus was 3616.36 mg/kg DW. The growth rate of long-term cell line was 3.5 folds after 18 days culture, but the content of resveratrol was low (21.79 mg/kg DW). By adding elicitor to medium, the content of resveratrol in long-term cells only up to 267.29 mg/kg DW. It was expressed that the productivity of resveratrol in long-term cell line was fast decrease.

並列關鍵字

plant cell culture ； resveratrol ； Vitis thunbergii

參考文獻

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