藏药柳茶愈伤组织诱导及褐化抑制探究
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摘要
本研究以藏药柳茶(Sibiraea angustata (Rehd.) Hand.-Mazz.)为研究材料,以MS为基本培养基,附加6-BA、NAA、2,4-D和KT等4种植物激素,采用正交试验研究不同激素配比对藏药愈伤组织诱导的影响,同时从消毒灭菌处理以及抗褐化剂类型的选择等方面进行了综合研究,以期为利用现代生物技术方法保护和利用藏药柳茶物种资源提供一种新的途径和方法。结果表明:经过75%酒精(0.5 min)+3%NaClO (1 min)+0.1%HgCl_2(5 min)混合消毒处理后的柳茶叶片与带腋芽茎段,其消毒效果最佳;与柳茶的叶片相比,带腋芽茎段的外植体在基础培养基(MS)中添加2 mg/L 6-BA及1 mg/L NAA后诱导出芽率最高,可达到86.67%,适合作为外植体进行组培快繁;在培养基中添加不同种类的抗褐化剂均可减轻外植体的褐化程度,但添加0.01 g/L AgNO_3后的抗褐化效果最佳且差异显著(p<0.05),可使柳茶叶片和带腋芽茎段的褐化率分别降至45.6%、14.4%。因此,经消毒处理、添加生长激素及抗褐化处理后的柳茶带腋芽茎段,其愈伤组织诱导率最高,适合作为柳茶的最佳外植体进行组培快繁。藏药柳茶愈伤组织诱导体系的建立将为青藏高原特有植物柳茶的种质资源保护和引种驯化以及关键功能基因的分子生物学研究提供材料和技术基础。
This study taking the stems and leaves of Tibetan medicine Sibiraea angustata(Rehd.) Hand.-Mazz. as the test material, using MS as the basic medium supplemented with 6-BA, NAA, 2,4-D and KT. The effects of different hormone concentrations and ratios on callus induction of Sibiraea angustata(Rehd.) Hand.-Mazz. were investigated by orthogonal design methods, the anti-browning agent type and treatment of disinfection sterilization and screening were carried out in this study. It provides a new way for protecting and utilizing Sibiraea angustata(Rehd.) Hand.-Mazz. species resources through the modern biotechnology. The result showed that the mixed treatment with 75% alcohol(0.5 min)+3% NaClO(1 min)+0.1% HgCl_2(5 min) was the best method for the leaves and stem with axillary bud. There were the highest induction rate with 86.67% of the stem with axillary buds,compared with the leaves as the explant, when using the basal medium(MS) with 2 mg/L 6-BA and 1 mg/L NAA in the study. There was a highly significant difference between the 0.01 g/L AgNO_3 and other kinds anti-browning agent(p<0.05), although all which could reduce the browning degree of the explants, and the browning rate of leaves and stem with axillary buds fell to 45.6%, 14.4%, respectively. Therefore, the best explant of Sibiraea angustata for rapid propagation was the stem with axillary buds treated with sterilization and growth hormone and antibrowning agent. The present study on the tissue culture of Sibiraea angustata(Rehd.) Hand.-Mazz. would not only provide material and technical basis for germplasm conservation and introduction domestication, but also help to research for molecular biology breeding of the key functional genes.
This study taking the stems and leaves of Tibetan medicine Sibiraea angustata(Rehd.) Hand.-Mazz. as the test material, using MS as the basic medium supplemented with 6-BA, NAA, 2,4-D and KT. The effects of different hormone concentrations and ratios on callus induction of Sibiraea angustata(Rehd.) Hand.-Mazz. were investigated by orthogonal design methods, the anti-browning agent type and treatment of disinfection sterilization and screening were carried out in this study. It provides a new way for protecting and utilizing Sibiraea angustata(Rehd.) Hand.-Mazz. species resources through the modern biotechnology. The result showed that the mixed treatment with 75% alcohol(0.5 min)+3% NaClO(1 min)+0.1% HgCl_2(5 min) was the best method for the leaves and stem with axillary bud. There were the highest induction rate with 86.67% of the stem with axillary buds,compared with the leaves as the explant, when using the basal medium(MS) with 2 mg/L 6-BA and 1 mg/L NAA in the study. There was a highly significant difference between the 0.01 g/L AgNO_3 and other kinds anti-browning agent(p<0.05), although all which could reduce the browning degree of the explants, and the browning rate of leaves and stem with axillary buds fell to 45.6%, 14.4%, respectively. Therefore, the best explant of Sibiraea angustata for rapid propagation was the stem with axillary buds treated with sterilization and growth hormone and antibrowning agent. The present study on the tissue culture of Sibiraea angustata(Rehd.) Hand.-Mazz. would not only provide material and technical basis for germplasm conservation and introduction domestication, but also help to research for molecular biology breeding of the key functional genes.
引文
An X.Y.,2015,Pharmacodynamics study and component analysis of two plants in Gansu Province,Thesis for M.S.,Lanzhou University,Supervisor:Yang Y.J.,pp.4-5(安小勇,2015,甘肃两种植物部分药效研究及成分分析,硕士学位论文,兰州大学,导师:杨永健,pp.4-5)
Cheng Y.,Hasiqi M.G.,Qin X.Z.,Tang X.Y.,Chen J.N.,Wang H.Y.,and Gao A.,2016,Effects of traditional Tibetan drug Liu Tea on proliferation and chemotherapeutic sensitivity of drug-resistant human gastric cancer cell BGC823/5-FU,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medica),41(4):603-608(程燕,哈斯其美格,覃小珍,唐祥友,陈健楠,王慧莹,高傲,2016,藏药柳茶水提物对胃癌耐药细胞BGC823/5-FU增殖及药物敏感性的影响,中国中药杂志,41(4):603-608)
Chen Y.,Shi X.Y.,Li H.L.,and Luo G.H.,2017,The analysis of chemical composition of volatile oil from leaves of Sibiraea angustata by GS-MS,Shipin Gongye Keji(Science and Technology of Food Industry),38(12):31-33(陈叶,石秀云,李海亮,罗光宏,2017,窄叶鲜卑花叶挥发油成分分析,食品工业科技,38(12):31-33)
Feng D.D.,Wang Y.,and Chen J.P.,2015,Research progress of browning in the plant tissue culture,Zhejiang Nongye Kexue(Acta Agriculturae Zhejiangensis),27(6):1108-1116(冯代弟,王燕,陈剑平,2015,植物组培褐化发生机制的研究进展,浙江农业学报,27(6):1108-1116)
He H.L.,Yang X.C.,Li D.D.,Yin C.L.,Li Y.X.,Zhou G.Y.,Zhang L.,and Liu Q.,2017,Stoichiometric characteristics of carbon,nitrogen and phosphorus of Sibiraea angustata shrub on the eastern Qinghai-Xizang Plateau,Zhiwu Shengtai Xuebao(Chinese Journal of Plant Ecology),41(1):126-135(贺合亮,阳小成,李丹丹,尹春英,黎云祥,周国英,张林,刘庆,2017,青藏高原东部窄叶鲜卑花碳、氮、磷化学计量特征,植物生态学报,41(1):126-135)
Pan Y.J.,Qin L.L.,Wu L.L.,Sun W.,Song Z.L.,Qin T.Y.,and Liu T.H.,2017,Progression of medicinal effects of Sibiraea angustata,Shijie Kexue Jishu Zhongyiyao Xiandaihua(Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology),(10):1737-1741(潘雅婧,秦灵灵,吴丽丽,孙文,宋紫临,秦田雨,刘铜华,2017,藏药柳茶的药用功效研究进展,世界科学技术-中医药现代化,(10):1737-1741)
Qi P.Y.,Fan W.J.,Hu X.,Liang H.Z.,Wu Y.Q.,and Xu B.Q.,2017,Callus induction and plant regeneration from stem explants of Campanumoea javanica B1,Zhejiang Nongye Xuebao(Acta Agriculturae Zhejiangensis),29(8):1313-1320(戚甫友,范伟军,胡秀,梁韩枝,吴永清,许炳强,2017,土党参茎段的愈伤组织诱导与植株再生,浙江农业学报,29(8):1313-1320)
Wang H.G.,Wu Y.,and Yue H.P.,2016,The effect of extracts Sibraea angustata on the expression of Mrp2 and Mrp6 of INH-induced hepatotoxicity in mice,Shizhen Guoyi Guoyao(Lishizhen Medicine and Materia Medica Research),27(11):2594-2595(王汉国,武燕,岳汉萍,2016,藏药柳茶提取物对异烟肼引起的肝损伤小鼠膜转运体Mrp2和Mrp6表达影响,时珍国医国药,27(11):2594-2595)
Wang Q.Y.,Tang J.N.,Liu Z.G.,Zhang M.R.,and Shen Y.M.,2017,Explant selecting and anti-browning of Magnolia sinostellata in tissue culture,Guangxi Zhiwu(Guihaia),37(9):1088-1095(王倩颖,唐佳妮,刘志高,张明如,申亚梅,2017,景宁木兰组织培养外植体选择与抗褐化研究,广西植物,37(9):1088-1095)
Wang X.,and Ma H.P.,2017,Advances in molecular evolution and main components of Sibraea angustata(Rehd.)Hand.-Mazz.,Zhongguo Minzu Minjian Yiyao(Chinese Journal of Ethnomedicine and Ethnopharmacy),26(16):56-58(王昕,马慧萍,2017,藏药柳茶分子进化特征与主要成分研究概况,中国民族民间医药,26(16):56-58)
Wang Z.W.,Xu X.H.,Chen X.T.,Yu S.S.,Liu H.D.,Lin L.G.,and Li B.,2014,Chemical constituents from the aerial part of Sibiraea angustata,Zhongyaocai(Journal of Chinese Medicinal Materials),37(1):57-60(王章伟,徐向红,陈笑天,庾石山,刘宏栋,林利光,李斌,2014,窄叶鲜卑花地上部分化学成分研究,中药材,37(1):57-60)
Wei Y.F.,2012,Preliminary study of Liucha on diabetic mice induced by alloxan and free fatty acids in rats with hyperlipidemia,Thesis for M.S.,Lanzhou University,Supervisor:Yang Y.J.,pp.9-10(卫阳飞,2012,柳茶对四氧嘧啶糖尿病小鼠降糖作用和对高脂血症大鼠游离脂肪酸影响的实验研究,硕士学位论文,兰州大学,导师:杨永健,pp.9-10)
Wu Y.,and Liu J.,2017,Intervention effect of Sibiraea angustata volatile oil on acute myocardial ischemic injury in mice,Zhongguo Xiandai Yingyong Yaoxue(Chinese Journal of Modern Applied Pharmacy),34(3):315-318(武燕,刘健,2017,藏药柳茶挥发油对小鼠急性心肌缺血损伤的干预作用,中国现代应用药学,34(3):315-318)
Xie Y.,Xia K.,Li M.,and Wu C.L.,2017,Effects of different angiosperms on callus induction and sterilization methods research,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(3):1029-1034(谢羽,夏凯,黎敏,伍春莲,2017,不同类被子植物组培快繁及其消毒方式的影响,分子植物育种,15(3):1029-1034)
Yao Z.Z,Liu D.H,,Chen X.T.,Li F.,Zhou X.,Pan L.L.,Wu Y.F.,Zhang D.M.,and Li B.,2018,Chemical constituents from the aerial part of Sibraea angustata,Zhongguo Yaoxue Zazhi(Chinese Pharmaceutical Journal),53(4):263-267(姚争争,刘德鸿,陈笑天,李斐,周翔,潘玲玲,吴亚芬,张东明,李斌,2018,窄叶鲜卑花地上部分化学成分研究,中国药学杂志,53(4):263-267)
Yang S.,Fang J.P.,and Wang Y.,2012,Study on influencing factors on browning rate of explants in tissue culture of Gymnadenia conopse,Guizhou Nongye Kexue(Guizhou Agricultural Sciences),40(1):22-25(杨爽,方江平,王勇,2012,手掌参组织培养中外植体褐化的影响因素研究,贵州农业科学,40(1):22-25)
Yuan Y.X.,Zhu L.J.,Wang J.P.,and Zhang P.,2014,Study on callus induction and browning prevention of Taxus chinensis var.mairei,Beifang Yuanyi(Northern Horticulture),(19):90-92(袁云香,朱丽君,王建鹏,张鹏,2014,南方红豆杉愈伤组织诱导及褐化防治研究,北方园艺,(19):90-92)
Zeng Q.F.,Wei X.,Chen X.,Chen G.J.,Ma P.J.,Wu J.H.,and Wang X.L.,2017,Optimisation and establishment of a high frequency regeneration system for Lolium multiflorum'Tetragold'and L.perenne'Four seasons',Caoye Kexue(Pratacultural Science),34(8):1649-1660(曾庆飞,韦鑫,陈锡,陈光洁,马培杰,吴佳海,王小利,2017,多花黑麦草特高和多年生黑麦草四季高频组培再生体系的优化与建立,草业科学,34(8):1649-1660)
Zhang D.J.,Yuan W.T.,Li M.T.,Liu M.C.,and Zhang Y.H.,2015,Correlation analysis between gene polymorphism of trnS-G sequence and content of salidrosides in Tibetan medi-cine of Rhodiola taohoensis S.H.Fu,Jiyinzuxue Yu Ying-yong Shengwuxue(Genomics and Applied Biology),34(1):130-135(张得钧,元王涛,李梦婷,刘明成,张育浩,2015,藏药洮河红景天trnS-G基因与红景天苷含量变异的相关性研究,基因组学与应用生物学,34(1):130-135)
Cheng Y.,Hasiqi M.G.,Qin X.Z.,Tang X.Y.,Chen J.N.,Wang H.Y.,and Gao A.,2016,Effects of traditional Tibetan drug Liu Tea on proliferation and chemotherapeutic sensitivity of drug-resistant human gastric cancer cell BGC823/5-FU,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medica),41(4):603-608(程燕,哈斯其美格,覃小珍,唐祥友,陈健楠,王慧莹,高傲,2016,藏药柳茶水提物对胃癌耐药细胞BGC823/5-FU增殖及药物敏感性的影响,中国中药杂志,41(4):603-608)
Chen Y.,Shi X.Y.,Li H.L.,and Luo G.H.,2017,The analysis of chemical composition of volatile oil from leaves of Sibiraea angustata by GS-MS,Shipin Gongye Keji(Science and Technology of Food Industry),38(12):31-33(陈叶,石秀云,李海亮,罗光宏,2017,窄叶鲜卑花叶挥发油成分分析,食品工业科技,38(12):31-33)
Feng D.D.,Wang Y.,and Chen J.P.,2015,Research progress of browning in the plant tissue culture,Zhejiang Nongye Kexue(Acta Agriculturae Zhejiangensis),27(6):1108-1116(冯代弟,王燕,陈剑平,2015,植物组培褐化发生机制的研究进展,浙江农业学报,27(6):1108-1116)
He H.L.,Yang X.C.,Li D.D.,Yin C.L.,Li Y.X.,Zhou G.Y.,Zhang L.,and Liu Q.,2017,Stoichiometric characteristics of carbon,nitrogen and phosphorus of Sibiraea angustata shrub on the eastern Qinghai-Xizang Plateau,Zhiwu Shengtai Xuebao(Chinese Journal of Plant Ecology),41(1):126-135(贺合亮,阳小成,李丹丹,尹春英,黎云祥,周国英,张林,刘庆,2017,青藏高原东部窄叶鲜卑花碳、氮、磷化学计量特征,植物生态学报,41(1):126-135)
Pan Y.J.,Qin L.L.,Wu L.L.,Sun W.,Song Z.L.,Qin T.Y.,and Liu T.H.,2017,Progression of medicinal effects of Sibiraea angustata,Shijie Kexue Jishu Zhongyiyao Xiandaihua(Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology),(10):1737-1741(潘雅婧,秦灵灵,吴丽丽,孙文,宋紫临,秦田雨,刘铜华,2017,藏药柳茶的药用功效研究进展,世界科学技术-中医药现代化,(10):1737-1741)
Qi P.Y.,Fan W.J.,Hu X.,Liang H.Z.,Wu Y.Q.,and Xu B.Q.,2017,Callus induction and plant regeneration from stem explants of Campanumoea javanica B1,Zhejiang Nongye Xuebao(Acta Agriculturae Zhejiangensis),29(8):1313-1320(戚甫友,范伟军,胡秀,梁韩枝,吴永清,许炳强,2017,土党参茎段的愈伤组织诱导与植株再生,浙江农业学报,29(8):1313-1320)
Wang H.G.,Wu Y.,and Yue H.P.,2016,The effect of extracts Sibraea angustata on the expression of Mrp2 and Mrp6 of INH-induced hepatotoxicity in mice,Shizhen Guoyi Guoyao(Lishizhen Medicine and Materia Medica Research),27(11):2594-2595(王汉国,武燕,岳汉萍,2016,藏药柳茶提取物对异烟肼引起的肝损伤小鼠膜转运体Mrp2和Mrp6表达影响,时珍国医国药,27(11):2594-2595)
Wang Q.Y.,Tang J.N.,Liu Z.G.,Zhang M.R.,and Shen Y.M.,2017,Explant selecting and anti-browning of Magnolia sinostellata in tissue culture,Guangxi Zhiwu(Guihaia),37(9):1088-1095(王倩颖,唐佳妮,刘志高,张明如,申亚梅,2017,景宁木兰组织培养外植体选择与抗褐化研究,广西植物,37(9):1088-1095)
Wang X.,and Ma H.P.,2017,Advances in molecular evolution and main components of Sibraea angustata(Rehd.)Hand.-Mazz.,Zhongguo Minzu Minjian Yiyao(Chinese Journal of Ethnomedicine and Ethnopharmacy),26(16):56-58(王昕,马慧萍,2017,藏药柳茶分子进化特征与主要成分研究概况,中国民族民间医药,26(16):56-58)
Wang Z.W.,Xu X.H.,Chen X.T.,Yu S.S.,Liu H.D.,Lin L.G.,and Li B.,2014,Chemical constituents from the aerial part of Sibiraea angustata,Zhongyaocai(Journal of Chinese Medicinal Materials),37(1):57-60(王章伟,徐向红,陈笑天,庾石山,刘宏栋,林利光,李斌,2014,窄叶鲜卑花地上部分化学成分研究,中药材,37(1):57-60)
Wei Y.F.,2012,Preliminary study of Liucha on diabetic mice induced by alloxan and free fatty acids in rats with hyperlipidemia,Thesis for M.S.,Lanzhou University,Supervisor:Yang Y.J.,pp.9-10(卫阳飞,2012,柳茶对四氧嘧啶糖尿病小鼠降糖作用和对高脂血症大鼠游离脂肪酸影响的实验研究,硕士学位论文,兰州大学,导师:杨永健,pp.9-10)
Wu Y.,and Liu J.,2017,Intervention effect of Sibiraea angustata volatile oil on acute myocardial ischemic injury in mice,Zhongguo Xiandai Yingyong Yaoxue(Chinese Journal of Modern Applied Pharmacy),34(3):315-318(武燕,刘健,2017,藏药柳茶挥发油对小鼠急性心肌缺血损伤的干预作用,中国现代应用药学,34(3):315-318)
Xie Y.,Xia K.,Li M.,and Wu C.L.,2017,Effects of different angiosperms on callus induction and sterilization methods research,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(3):1029-1034(谢羽,夏凯,黎敏,伍春莲,2017,不同类被子植物组培快繁及其消毒方式的影响,分子植物育种,15(3):1029-1034)
Yao Z.Z,Liu D.H,,Chen X.T.,Li F.,Zhou X.,Pan L.L.,Wu Y.F.,Zhang D.M.,and Li B.,2018,Chemical constituents from the aerial part of Sibraea angustata,Zhongguo Yaoxue Zazhi(Chinese Pharmaceutical Journal),53(4):263-267(姚争争,刘德鸿,陈笑天,李斐,周翔,潘玲玲,吴亚芬,张东明,李斌,2018,窄叶鲜卑花地上部分化学成分研究,中国药学杂志,53(4):263-267)
Yang S.,Fang J.P.,and Wang Y.,2012,Study on influencing factors on browning rate of explants in tissue culture of Gymnadenia conopse,Guizhou Nongye Kexue(Guizhou Agricultural Sciences),40(1):22-25(杨爽,方江平,王勇,2012,手掌参组织培养中外植体褐化的影响因素研究,贵州农业科学,40(1):22-25)
Yuan Y.X.,Zhu L.J.,Wang J.P.,and Zhang P.,2014,Study on callus induction and browning prevention of Taxus chinensis var.mairei,Beifang Yuanyi(Northern Horticulture),(19):90-92(袁云香,朱丽君,王建鹏,张鹏,2014,南方红豆杉愈伤组织诱导及褐化防治研究,北方园艺,(19):90-92)
Zeng Q.F.,Wei X.,Chen X.,Chen G.J.,Ma P.J.,Wu J.H.,and Wang X.L.,2017,Optimisation and establishment of a high frequency regeneration system for Lolium multiflorum'Tetragold'and L.perenne'Four seasons',Caoye Kexue(Pratacultural Science),34(8):1649-1660(曾庆飞,韦鑫,陈锡,陈光洁,马培杰,吴佳海,王小利,2017,多花黑麦草特高和多年生黑麦草四季高频组培再生体系的优化与建立,草业科学,34(8):1649-1660)
Zhang D.J.,Yuan W.T.,Li M.T.,Liu M.C.,and Zhang Y.H.,2015,Correlation analysis between gene polymorphism of trnS-G sequence and content of salidrosides in Tibetan medi-cine of Rhodiola taohoensis S.H.Fu,Jiyinzuxue Yu Ying-yong Shengwuxue(Genomics and Applied Biology),34(1):130-135(张得钧,元王涛,李梦婷,刘明成,张育浩,2015,藏药洮河红景天trnS-G基因与红景天苷含量变异的相关性研究,基因组学与应用生物学,34(1):130-135)