金银花愈伤组织诱导及绿原酸含量测定
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摘要
优化金银花愈伤组织诱导主要条件,建立愈伤组织培养体系并测定其绿原酸含量。结果表明,金银花芽为最佳的诱导外植体,经过70%酒精30 s、5%的次氯酸钠10 min消毒处理,接种于激素配比为NAA 1.5 mg/L+2,4-D 1.0 mg/L+KT 0.75 mg/L+6-BA 0.15 mg/L的MS培养基中,暗培养9 d后再进行光、暗交替培养,可诱导出淡黄色,疏松、生长旺盛的愈伤组织;愈伤组织生长曲线呈S型,24 d生长量为(21.62±0.54)g/瓶;通过高效液相检测,愈伤组织每100 g样品含有绿原酸57 mg。经验证金银花愈伤组织诱导条件稳定,含一定的绿原酸为进一步建立细胞悬浮培养体系高效生物积累金银花细胞及绿原酸功能成分奠定基础。
Main conditions for Lonicera japonica Thunb callus induction was optimized,a callus culture system was established and its chlorogenic acid content was determined. The results showed that L. japonica Thunb bud was the best induction explant; it was sterilized in 70% alcohol 10 min and then in 5% sodium hypochlorite 30 s,then transferred onto the medium of MS+NAA 1.5 mg / L+ 2,4-D 1.0 mg / L+ KT 0.75 mg / L+6-BA 0.15 mg / L,cultrured in dark for 9 days before light and dark alternate culture,friable,vigorous and light yellow coloured callus could be induced.The callus growth curve was in the shape of "S",and the highest biomass yield was 21.62±0.54 g / bottle at the 24 th day; the chlorogenic acid content in callus was 57 mg / 100 g,according to the high performance liquid chromatogram detection.The optimized induction technology is stable;rich chlorogenic acid offered a reliable theory basis for further establishment of cell suspension culture system for bioaccumulation cells and chlorogenic acid functional ingredients of L.japonica Thunb.
Main conditions for Lonicera japonica Thunb callus induction was optimized,a callus culture system was established and its chlorogenic acid content was determined. The results showed that L. japonica Thunb bud was the best induction explant; it was sterilized in 70% alcohol 10 min and then in 5% sodium hypochlorite 30 s,then transferred onto the medium of MS+NAA 1.5 mg / L+ 2,4-D 1.0 mg / L+ KT 0.75 mg / L+6-BA 0.15 mg / L,cultrured in dark for 9 days before light and dark alternate culture,friable,vigorous and light yellow coloured callus could be induced.The callus growth curve was in the shape of "S",and the highest biomass yield was 21.62±0.54 g / bottle at the 24 th day; the chlorogenic acid content in callus was 57 mg / 100 g,according to the high performance liquid chromatogram detection.The optimized induction technology is stable;rich chlorogenic acid offered a reliable theory basis for further establishment of cell suspension culture system for bioaccumulation cells and chlorogenic acid functional ingredients of L.japonica Thunb.
引文
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[11]Ali M,Abbasi B H,Ihsan-ul-haq.Production of commercially important secondary metabolites and antioxidant activity in cell suspension cultures of Artemisia absinthium L[J].Industrial Crops&Products,2013,49(8):400-406.
[12]文明玲,乐正碧,徐茜,等.金银花组织培养外植体灭菌方法探索[J].陕西农业科学,2011(2):50-51.Weng M L,Le Z B,Xu Q,et al.Explore explant sterilization method of honeysuckle tissue culture[J].Shaanxi Agricultural Sciences,2011(2):50-51.
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[14]任敏,赵红艳.金银花组织培养无菌体系的建立[J].新乡学院学报(自然科学版),2009,26(3):49-51.Ren M,Zhao H Y.Tissue culture and rapid propagation of Lonicera Japonica Thunb.[J].Journal of Xinxiang University(Natural Science Edition),2009,26(3):49-51.
[15]马萌萌.金银花新品种“忍冬一号”光合生理特性与组培快繁技术研究[D].山东中医药大学,2010:27-28.Ma M M.Research of physiological property,tissue culture and rapid propagation technology on a new variety of Lonicera japonica Thunb.yihao[D].Shandong Traditional Chinese Medicine University,2010:27-28.
[16]单广福,张晓丽,李明军,等.金银花愈伤组织的诱导及继代培养研究[J].北方园艺,2013(5):106-109.Shan G F,Zhang X L,Li M J,et al.Research of honeysuckle callus induction and successive transfer culture[J].Northern Hourtculture,2013(5):106-109.
[17]孙镇,袁丽红,吴频梅.诱导子对藏红花悬浮培养细胞生产藏红花色素的影响[J].生物加工过程,2013,11(3):18-22.Sun Z,Yuan L H,Wu B M.Effects of elicitors on saffron pigment production in cell suspension cultures of crocus sativus L.[J].Chinese Journal of Bioprocess Engineering,2013,11(3):18-22.
[2]葛冰,卢向阳,易克,等.金银花活性成分、药理作用及其应用[J].中国野生植物资源,2004,23(5):13-15.Ge B,Lu X Y,Yi K,et al.The active constituent and pharmacological action of flos lonicerae and its application[J].China’s Wild Plant Resources,2004,23(5):13-15.
[3]Rumalla C S,Avula B,Zhao J P,et al.Quantitative determination of phenolic acids in Lonicera japonica Thunb using high performance thin layer chromatography[J].Journal of Liquid Chromatography&Related Technologies,2011,34(1):38-47.
[4]Xiong J H,Li S C,Wang W J,et al.Screening and identification of the antibacterial bioactive compounds from Lonicera japonica Thunb.leaves[J].Food Chemistry,2013,138(05):327-333.
[5]李世传,熊建华,罗秋水,等.不同干燥方法对金银花叶成分和抑菌效果的影响[J].中国食品学报,2012,12(12):78-83.Li S C,Xiong J H,Luo Q S,et al.The effect of different drying methods on honeysuckle leaves’components and bacteriostatic effect[J].Journal of Chinese Institute of Food Science and Technology,2012,12(12):78-83.
[6]Sun C H,Teng Y,Li G Z,et al.Metabonomics study of the protective effects of Lonicera japonica extract on acute liver injury in dimethylnitrosamine treated rats[J].Journal of Pharmaceutical and Biomedical Analysis,2010,53(1):98-102
[7]Rahman A,Kang S C.In vitro control of food-borne and food spoilage bacteria by essential oil and ethanol extracts of Lonicera japonica Thunb.[J].Food Chemistry,2009,116(3):670-675
[8]崔婷婷,王超,单长松,等.金银花开发利用的研究进展[J].饮料工业,2014,17(6):55-60.Cui T T,Wang C,Shan C S,et al.Review on the development and utilization of honeysuckle[J].The Beverage Industry,2014,17(6):55-60.
[9]Maneechai S,De-Eknamkul W,Umehara K,et al.Flavonoid and stilbenoid production in callus cultures of Artocarpus lakoocha[J].Phytochemistry,2012,81(09):42-49.
[10]Giri L,Dhyani P,Rawat S,et al.In vitro production of phenolic compounds and antioxidant activity in callus suspension cultures of Habenaria edgeworthii:a rare Himalayan medicinal orchid[J].Industrial Crops&Products,2012,39(9):1-6.
[11]Ali M,Abbasi B H,Ihsan-ul-haq.Production of commercially important secondary metabolites and antioxidant activity in cell suspension cultures of Artemisia absinthium L[J].Industrial Crops&Products,2013,49(8):400-406.
[12]文明玲,乐正碧,徐茜,等.金银花组织培养外植体灭菌方法探索[J].陕西农业科学,2011(2):50-51.Weng M L,Le Z B,Xu Q,et al.Explore explant sterilization method of honeysuckle tissue culture[J].Shaanxi Agricultural Sciences,2011(2):50-51.
[13]赵贤慧,刘庆华,张德锋.红金银花组织培养外植体灭菌的研究[J].江苏林业科技,2007,34(1):23-25.Zhao X H,Liu Q H,Zhang D F.Sterilization of the explant of Lonicera japonica var.chinensis in vitro culture[J].Journal of Jiangsu Forestry Science and&Technology,2007,34(1):23-25.
[14]任敏,赵红艳.金银花组织培养无菌体系的建立[J].新乡学院学报(自然科学版),2009,26(3):49-51.Ren M,Zhao H Y.Tissue culture and rapid propagation of Lonicera Japonica Thunb.[J].Journal of Xinxiang University(Natural Science Edition),2009,26(3):49-51.
[15]马萌萌.金银花新品种“忍冬一号”光合生理特性与组培快繁技术研究[D].山东中医药大学,2010:27-28.Ma M M.Research of physiological property,tissue culture and rapid propagation technology on a new variety of Lonicera japonica Thunb.yihao[D].Shandong Traditional Chinese Medicine University,2010:27-28.
[16]单广福,张晓丽,李明军,等.金银花愈伤组织的诱导及继代培养研究[J].北方园艺,2013(5):106-109.Shan G F,Zhang X L,Li M J,et al.Research of honeysuckle callus induction and successive transfer culture[J].Northern Hourtculture,2013(5):106-109.
[17]孙镇,袁丽红,吴频梅.诱导子对藏红花悬浮培养细胞生产藏红花色素的影响[J].生物加工过程,2013,11(3):18-22.Sun Z,Yuan L H,Wu B M.Effects of elicitors on saffron pigment production in cell suspension cultures of crocus sativus L.[J].Chinese Journal of Bioprocess Engineering,2013,11(3):18-22.