黑荆树愈伤组织培养及其原花色素的分析

doi:10.3969/j.issn.0253-2417.2016.03.008

摘要/Abstract

摘要： 以黑荆树叶片为外植体,在MS培养基中添加不同质量浓度的细胞分裂素6-苄氨基嘌呤(6-BA)和细胞生长素2,4-二氯苯氧乙酸(2,4-D),诱导培养出愈伤组织,采用香草醛-硫酸法和Folin-Ciocalteu法测定了不同条件处理下愈伤组织中原花色素和总多酚的含量,并用电喷雾质谱(二级质谱)法对愈伤组织中原花色素组成进行定性分析。研究结果表明:添加0.25 mg/L 6-BA和 2.0 mg/L 2,4-D的培养基中,黑荆树愈伤组织诱导率及生长状况较佳,且含总多酚和原花色素分别为20.19和11.10 mg/g,明显高于其他处理,适合进行增殖培养;黑荆树愈伤组织主要由单体原花色素及低聚原花色素组成,其中以单体和二聚体居多,还有微量三聚体。

关键词: 黑荆树, 愈伤组织, 原花色素, 二级质谱

Abstract: This paper studied proanthocyanidins in callus of Acacia mearnsii De Wilde. Callus were obtained by inoculating leaf explants on MS medium, and supplemented with the combination of 6-BA and 2,4-D. The content of total phenolic compounds and proanthocyanidins in the 12 different treatments were tested by Folin-Ciocalteu assay and vanillin assay, respectively. Proanthocyanidins in callus were characterized by ESI-MS. The results showed that the leaf explants of A.mearnsii treated with 0.25 mg/L 6-BA in combination with 2.0 mg/L 2,4-D resulted in a higher rate of callus formation, better growth and maximum content of total phenolic compounds 20.19 mg/g and proanthocyanidins 11.10 mg/g, which is suitable for proliferation culture. We detected monomer, dimer and trace trimer in callus of A.mearnsii, too.

Key words: Acacia mearnsii De Wilde, callus, proanthocyanidin, ESI-MS²

中图分类号:

TQ35

徐玲玲, 杨阳, 王飞. 黑荆树愈伤组织培养及其原花色素的分析[J]. 林产化学与工业, 2016, 36(3): 53-59.

XU Ling-ling, YANG Yang, WANG Fei. Callus Culture of Acacia mearnsii De Wilde and Analysis on Proanthocyanidin[J]. Chemistry and Industry of Forest Products, 2016, 36(3): 53-59.

参考文献

[1] 任华东,姚小华,康文玲,等.黑荆树种源和家系的遗传变异与早期选择[J].林业科学,2010,46(3):153-160. REN Hua-dong,YAO Xiao-hua,KANG Wen-ling,et al.Genetic variation and early selection of provenances and families of Acacia mearnsii[J].Scientia Silvae Sinicae,2010,46(3):153-160.
[2] 张加研,付惠,刘祥义.黑荆树皮中抗氧化性物质化学成分研究初探[J].林产化工通讯,2004,38(2):14-17. ZHANG Jia-yan,FU Hui,LIU Xiang-yi.A preliminary study on oxidation resistance of the bark extract from Acacia mearnsii De Wilde[J].Journal of Chemical Industry of Forest Products,2004,38(2):14-17.
[3] 孙达旺.植物单宁化学[M].北京:中国林业出版社,1992. SUN Da-wang. Plant Tannins Chemical[M].Beijing:China Forestry Publishing House,1992.
[4] 国植,徐莉.原花青素:具有广阔发展前景的植物药[J].国外医药:植物药分册,1996,11(5):196-204. GUO Zhi,XU Li.Procyanidins:A kind of plant medicine with a broad prospects for development[J].Foreign Medical Science,1996,11(5):196-204.
[5] 曲均革.葡萄细胞培养生产花青素不稳定性研究[D].大连:中国科学院化学物理研究所博士学位论文,2006. QU Jun-ge.Studies on the instability of anthocyanin accumulation insuspension cultures of Vitis vinifera[D].Dalian:Doctoral Dissertation of Institute of Chemical Physics,Chinese Academy of Sciences,2006.
[6] CURTIN M E.Harvesting profitable products from plant tissue culture[J].Nature Biotechnology,1983,1(8):649-659.
[7] DICOSMO F,MISAWA M.Plant cell and tissue culture:Alternatives for metabolite production[J].Biotechnology Advances,1995,13(3):425-453.
[8] TABATA H.Paclitaxel Production by Plant-cell-culture Technology[M].Biomanufacturing.Springer Berlin Heidelberg,2004:1-23.
[9] 王亚琴,朱媛.杜仲细胞悬浮培养生产桃叶珊瑚甙的研究[J].广西植物,2007,27(2):236-239. WANG Ya-qin,ZHU Yuan.Studies on the cell suspension culture of Eucommia ulmoides Oliv.and its metabolite-aucubin[J].Guihaia,2007,27(2):236-239.
[10] SCHRALL R,BECKER H.Production of catechins and oligomeric proanthocyanidins in tissue and suspension cultures of Crataegus monogyna,C.oxyacantha and Ginkgo biloba (author's transl)[J].Planta Medica,1977,32(4):297.
[11] NAWA Y,ASANO S,MOTOORI S,et al.Production of anthocyanins,carotenoids,and proanthocyanidins by cultured cells of rabbiteye blueberry (Vaccinium ashei Reade)[J].Bioscience,biotechnology,and biochemistry,1993,57(5):770-774.
[12] 赖呈纯,范丽华,黄贤贵,等.刺葡萄幼胚愈伤组织诱导及其高产原花青素细胞系筛选[J].植物生理学报,2014,50(11):1683-1691. LAI Cheng-chun,FAN Li-hua,HUANG Xian-gui,et al.Callus Induction in brier grape (Vitis davidii Foëx) from immature embryos and screening of cell lines with high-production of oligomeric proanthocyanidins[J].Acta Phytophysiologica Sinica,2014,50(11):1683-1691.
[13] QUOIRIN M,BITTENCOURT J M,ZANETTE F,et al.Effect of growth regulators on indirect organogenesis of Acacia mearnsii tissues cultured in vitro[J].Revista Brasileira de Fisiologia Vegetal,1998,10(2):101-105.
[14] BECK S L,DUNLOP R,VAN STADEN J.Micropropagation of Acacia mearnsii from ex vitro material[J].Plant growth regulation,1998,26(3):143-148.
[15] BECK S L,BUNLOP R,VAN STADEN J.Meristem culture of Acacia mearnsii[J].Plant Growth Regulation,2000,32(1):49-58.
[16] 王敬文,蒋晶.黑荆树悬浮单细胞低温驯化[J].林业科学研究,1989,2(5):442-445. WANG Jing-wen,JIANG Jing.Cold acclimation of suspension cultures of Acacia mearnsii Wild[J].Forest Research,1989,2(5):442-446.
[17] 张庆华,黄文銮,蔡丽萍,等.黑荆树组织培养繁殖技术的研究[J].经济林研究,1993,11(1):33-36. ZHANG Qing-hua,HUANG Wen-luan,CAI Li-ping,et al.Tissue culture techniques for mearns acacia[J].Economic Forest Researches,1993,11(1):33-36.
[18] 龚峥,翟应昌,周志坚.黑荆树愈伤组织和细胞悬浮培养试验[J].广东林业科技,1993,1:11-15. GONG Zheng,ZHAI Ying-chang,ZHOU Zhi-jian.Black wattle callus and cell suspension culture experiment[J].Guangdong Forestry Science and Technology,1993(1):11-15.
[19] 唐佳佳,尚旭岚,洑香香.黑荆树愈伤组织诱导,增殖与分化[J].中南林业科技大学学报,2014,34(9):38-43. TANG Jia-jia,SHANG Xu-lan,FU Xiang-xiang.Induction,proliferation,and differentiation of Acacia mearnsii callus[J].Journal of Central South University of Forestry & Technology,2014,34(9):38-43.
[20] 唐佳佳,尚旭岚,洑香香.黑荆树不同外植体愈伤组织诱导与芽分化的研究[J].江苏林业科技,2014,41(2):6-10. TANG Jia-jia,SHANG Xu-lan,FU Xiang-xiang.Callus induction and differentiation with diverse explants of Acacia mearnsii[J].Journal of Jiangsu Forestry Science & Technology,2014,41(2):6-10.
[21] 鲍俊竹,陈月坤,徐桂花.测定葡萄籽提取物中原花青素含量的方法[J].农业科学研究,2005,26(1):43-45. BAO Jun-zhu,CHEN Yue-kun,XU Gui-hua.Primary study on the determination methods of proanthocyanidin content of grape seed extract[J].Journal of Agricultural Sciences,2005,26(1):43-45.
[22] SINGLETON V L,ORTHOFER R,LAMUELA-RAVENTOS R M.Analysis of total phenols and other oxidationsubstrates andantioxidants by means of folin-ciocalteu reagent[J].Methods in enzymology,1999,299:152-178.
[23] 杜亚填,陈建华,许建宇,等.植物生长调节剂对南方红豆杉愈伤组织培养和紫杉醇合成的影响[J].天然产物研究与开发,2006,18(4):569-576. DU Ya-tian,CHEN Jian-hua,XU Jian-yu,et al.Influence of plant growth regulator on Taxus chinensis var.mairei callus culture and biosynthesis of taxol[J].Natural Product Research and Development,2006,18(4):569-576.
[24] 蔡国琴,李国珍,叶和春,等.Ri质粒转化的青蒿发根培养及青蒿素的生物合成[J].生物工程学报,1995,11(4):315-320. CAI Guo-qin,LI Guo-zhen,YE He-chun,et al.Hairy root culture of Artemisia annua L.by Riplasmid transformation and biosynthesis of artemisinin[J].Chinese Journal of Biotechnology,1995,11(4):315-320,408.
[25] VENTER P B,SENEKAL N D,KEMP G,et al.Analysis of commercial proanthocyanidins.Part 3:The chemical composition of wattle (Acacia mearnsii) bark extract[J].Phytochemistry,2012,83:153-167.]