诺丽茎段愈伤组织诱导优化及细胞悬浮系的建立
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摘要
为获取诺丽茎段中的次生代谢物并为建立遗传转化体系奠定基础,以诺丽茎段(无腋芽)为外植体诱导愈伤组织,并建立细胞悬浮系,对影响愈伤组织的诱导及细胞悬浮系的因子进行了研究。结果表明:愈伤组织诱导的最优培养基是MS+1.0mg/L6-BA+0.1mg/L2,4-D;悬浮培养的最佳培养基为MS+1.0mg/L6-BA+0.1mg/L2,4-D+3%蔗糖,pH为5.85,当初始接种量为37.5g/L、摇床转速为110r/min且(25±2)℃暗培养时,悬浮细胞生长良好,生长速率最大;诺丽茎段悬浮细胞生长曲线呈"S"型,最适继代周期为12–20 d;培养过程中,培养基的pH呈先下降后缓慢升高的变化趋势,诺丽茎段愈伤组织悬浮细胞培养的最适pH在4.5–5.0之间。文中成功建立了以诺丽茎段为外植体的稳定的细胞悬浮系。
The aim of the study was to obtain the secondary metabolites in the stem segment of noni and to establish genetic transformation system. The stem segments(no axillary buds) of noni were used as explants to induce the callus, and then to establish the cell suspension system. The factors affecting callus induction and cell suspension were studied. The results showed that the optimal culture medium for induction was MS with 1.0 mg/L 6-Benzylaminopurine(6-BA) and 0.1 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D), and the optimum culture medium for suspension was MS with 1.0 mg/L 6-BA and 0.1 mg/L 2,4-D, 3% sucrose and the pH of 5.85, with the initial inoculation amount of 37.5 g/L, and the speed of 110 r/min and 25±2 ℃ applying darkness culture. The suspension cells grew well and showed the maximum growth rate. The growth curve of the suspension cells from the stem segment of noni was in "S-typed" trend, and it should be transformed to the fresh medium between 12 and 20 d. During the culture, the pH of the culture medium decreased and then slowly increased, and the optimum pH for the suspension cells culture of callus from noni's stem segments was 4.5–5.0. In this study, the stable cell suspension system of the stem segment of noni was successfully established.
The aim of the study was to obtain the secondary metabolites in the stem segment of noni and to establish genetic transformation system. The stem segments(no axillary buds) of noni were used as explants to induce the callus, and then to establish the cell suspension system. The factors affecting callus induction and cell suspension were studied. The results showed that the optimal culture medium for induction was MS with 1.0 mg/L 6-Benzylaminopurine(6-BA) and 0.1 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D), and the optimum culture medium for suspension was MS with 1.0 mg/L 6-BA and 0.1 mg/L 2,4-D, 3% sucrose and the pH of 5.85, with the initial inoculation amount of 37.5 g/L, and the speed of 110 r/min and 25±2 ℃ applying darkness culture. The suspension cells grew well and showed the maximum growth rate. The growth curve of the suspension cells from the stem segment of noni was in "S-typed" trend, and it should be transformed to the fresh medium between 12 and 20 d. During the culture, the pH of the culture medium decreased and then slowly increased, and the optimum pH for the suspension cells culture of callus from noni's stem segments was 4.5–5.0. In this study, the stable cell suspension system of the stem segment of noni was successfully established.
引文
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[6]Hirazumi A,Furusawa E.An immunomodulatory polysaccharide-rich substance from the fruit juice of Morinda citrifolia(noni)with antitumour activity.Phytother Res,1999,13(5):380-387.
[7]Wu T,Lan ZQ.Genetic relationship of Morinda citrifolia germplasms by ISSR.J Tropl Subtrop Bot,2014,22(6):617-623(in Chinese).吴田,蓝增全.基于ISSR技术研究诺丽种质资源的亲缘关系.热带亚热带植物学报,2014,22(6):617-623.
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[12]Wang GL,Shi RF,Fang HJ.Effects of medium and culture conditions on polysaccharide synthesis by suspension cell culture of Gardenia jasminoides Eills.Chin J Biotech,2001,17(6):688-692(in Chinese).王关林,石若夫,方宏筠.培养基和培养条件对栀子悬浮细胞合成多糖的影响.生物工程学报,2001,17(6):688-692.
[13]Lv DX,Qu CF.The effects that the regulating agenr for she plants growth takes to callus culfuring.Northern Horticult,2004(5):68-69(in Chinese).吕冬霞,曲长福.植物生长调节剂对愈伤组织培养的影响.北方园艺,2004(5):68-69.
[14]Zhao JP,Yang SS.Establishment of cell suspension culture system for Taxus media.J Northwest A&FUniv:Nat Sci Ed,2014,42(1):189-195(in Chinese).赵继鹏,杨淑慎.曼地亚红豆杉细胞悬浮培养体系的建立.西北农林科技大学学报:自然科学版,2014,42(1):189-195.
[15]Gao Y,Sun MD,Xu QZ,et al.Establishment of callus induction and cell suspension culture system of Sophora alopecuroides.Jiangsu Agricl Sci,2017,45(14):27-31(in Chinese).高媛,孙牧笛,徐全智,等.苦豆子愈伤组织诱导及细胞悬浮培养体系的建立.江苏农业科学,2017,45(14):27-31.
[16]Yang JL,Gui YL,Guo ZC.Studies on kinetics of somatic embryo suspension culture in Picea meyeri Rehd.et Wils.Chin J Biotech,2000,16(2):218-220(in Chinese).杨金玲,桂耀林,郭仲琛.白杄体细胞胚悬浮培养的动力学研究.生物工程学报,2000,16(2):218-220.
[17]Yang YG,Gui YL,Tang W,et al.Observation on differentiation potential and chromosome stability of callus in subcultures of Picea wilsonii.Acta Botan Sin,1994,36(12):934-939(in Chinese).杨映根,桂耀林,唐巍,等.青杄愈伤组织在继代培养中的分化能力及染色体稳定性研究.植物学报,1994,36(12):934-939.
[18]Salaj T,BlehováA,Salaj J.Embryogenic suspension cultures of Pinus nigra Arn:Growth parameters and maturation ability.Acta Physiol Plant,2007,29(3):225-231.
[19]Yu B,Liu JM,Liu XR,et al.An efficient system of embryogenic cell suspension cultures and plant regeneration in Spathiphyllum cannifolium.Acta Horticultu Sin,2015,42(4):721-738(in Chinese).于波,刘金梅,刘晓荣,等.白鹤芋胚性细胞悬浮培养和高效植株再生体系的建立.园艺学报,2015,42(4):721-738.
[20]Mukherjee S,Ghosh B,Jha S.Establishment of forskolin yielding transfomed cell suspension cultures of Coleus forskohlii as controlledby different factors.J Biotechnol,2000,76(1):73-81.
[21]Chen YQ,Zhu WH,Wu YQ,et al.Effects of fungal elicitor on taxol prodction in suspension cells of Taxus yunnanensis.Chin J Biotech,1999,15(4):522-524(in Chinese).陈永勤,朱蔚华,吴蕴祺,等.几种真菌诱导子对云南红豆杉细胞产生紫杉醇的影响.生物工程学报,1999,15(4):522-524.
[22]Zhang CP,Li C,Yuan YJ.Effects of fungal elicitor on secondary metabolism of cell suspension culture of Taxus chinensis var.mairei.J Chem Ind Eng(China),2002,53(5):498-502(in Chinese).张长平,李春,元英进.真菌诱导子对悬浮培养南方红豆杉细胞次生代谢的影响.化工学报,2002,53(5):498-502.
[23]Sun Z,Yuan LH,Wu PM.Effects of elicitors on saffron pigment production in cell suspension cultures of Crocus sativus L..Chin J Bioprs Eng,2013,11(3):18-23(in Chinese).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.生物加工过程,2013,11(3):18-23.
[24]Dong YL,Pan XW.Effects of copper ion induction elicitation on camptothecin biosynthesis in cell suspension culture of campototheca acuminata decaisne.J Anhui Agric Sci,2010,38(23):12457-12459(in Chinese).董妍玲,潘学武.Cu2+诱导刺激对喜树悬浮培养细胞喜树碱生物合成的影响.安徽农业科学,2010,38(23):12457-12459.
[25]Almagro L,García-Pérez P,Belchí-Navarro S,et al.New strategies for the use of Linum usitatissimum cell factories for the production of bioactive compounds.Plant Physiol Biochem,2015,99:73-78.
[26]Zhao J,Zhu W H,Hu Q.Enhanced catharanthine production in Catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors.Enzyme Microb Technol,2001,28(7/8):673-681.
[2]Yang XB.Hainan Plants Directory.Beijing:Science Press,2013:340-341(in Chinese).杨小波.海南植物名录.北京:科学出版社,2013:340-341.
[3]Chen XQ,Cui HB,Dai LK,et al.Flora of China:19.Beijing:Science Press,2011:180-187(in Chinese).陈心启,崔鸿宾,戴伦凯,等.中国植物志:19卷.北京:科学出版社,2011:180-187.
[4]Jiang JW.Dictionary of Medicinal Plants.Tianjin:Science and Technology Press,2005:875-972(in Chinese).江纪武.药用植物辞典.天津:科学技术出版社,2005:875-972.
[5]Dixon AR,Mcmillen H,Etkin NL.Ferment this:the transformation of noni,a traditional polynesian medicine(Morinda citrifolia,Rubiaceae).Econom Bot,1999,53(1):51-68.
[6]Hirazumi A,Furusawa E.An immunomodulatory polysaccharide-rich substance from the fruit juice of Morinda citrifolia(noni)with antitumour activity.Phytother Res,1999,13(5):380-387.
[7]Wu T,Lan ZQ.Genetic relationship of Morinda citrifolia germplasms by ISSR.J Tropl Subtrop Bot,2014,22(6):617-623(in Chinese).吴田,蓝增全.基于ISSR技术研究诺丽种质资源的亲缘关系.热带亚热带植物学报,2014,22(6):617-623.
[8]Huang Q,He WJ,Ye BY,et al.Studies on the micropropagation of noni(Morinda citrifolia L.).JFujian Norm Univ:Nat Sci Ed,2007,23(1):87-90(in Chinese).黄骐,何文锦,叶冰莹,等.诺丽(Morinda citrifolia L.)离体快速繁殖研究.福建师范大学学报:自然科学版,2007,23(1):87-90.
[9]Wu T,Lan ZQ.Study on culture in vitro of noni(Morinda citrifolia Linn.).J Shandong Agric Univ:Nat Sci,2011,42(2):179-182(in Chinese).吴田,蓝增全.诺丽离体培养研究.山东农业大学学报:自然科学版,2011,42(2):179-182.
[10]Pan XQ,Wu T,Lan ZQ,et al.Regeneration of stem segments of noni(Morinda citrifolia Linn.).JNortheast For Univ,2014,42(8):20-24(in Chinese).潘晓晴,吴田,蓝增全,等.诺丽茎段的离体再生.东北林业大学学报,2014,42(8):20-24.
[11]Zhang ZQ,Li YC.Study on callus induction and cell suspension culture of Morinda citrifolia.Guangdong Agric Sci,2014,41(4):108-112(in Chinese).张志强,李永成.海巴戟天愈伤组织诱导及细胞悬浮培养试验.广东农业科学,2014,41(4):108-112.
[12]Wang GL,Shi RF,Fang HJ.Effects of medium and culture conditions on polysaccharide synthesis by suspension cell culture of Gardenia jasminoides Eills.Chin J Biotech,2001,17(6):688-692(in Chinese).王关林,石若夫,方宏筠.培养基和培养条件对栀子悬浮细胞合成多糖的影响.生物工程学报,2001,17(6):688-692.
[13]Lv DX,Qu CF.The effects that the regulating agenr for she plants growth takes to callus culfuring.Northern Horticult,2004(5):68-69(in Chinese).吕冬霞,曲长福.植物生长调节剂对愈伤组织培养的影响.北方园艺,2004(5):68-69.
[14]Zhao JP,Yang SS.Establishment of cell suspension culture system for Taxus media.J Northwest A&FUniv:Nat Sci Ed,2014,42(1):189-195(in Chinese).赵继鹏,杨淑慎.曼地亚红豆杉细胞悬浮培养体系的建立.西北农林科技大学学报:自然科学版,2014,42(1):189-195.
[15]Gao Y,Sun MD,Xu QZ,et al.Establishment of callus induction and cell suspension culture system of Sophora alopecuroides.Jiangsu Agricl Sci,2017,45(14):27-31(in Chinese).高媛,孙牧笛,徐全智,等.苦豆子愈伤组织诱导及细胞悬浮培养体系的建立.江苏农业科学,2017,45(14):27-31.
[16]Yang JL,Gui YL,Guo ZC.Studies on kinetics of somatic embryo suspension culture in Picea meyeri Rehd.et Wils.Chin J Biotech,2000,16(2):218-220(in Chinese).杨金玲,桂耀林,郭仲琛.白杄体细胞胚悬浮培养的动力学研究.生物工程学报,2000,16(2):218-220.
[17]Yang YG,Gui YL,Tang W,et al.Observation on differentiation potential and chromosome stability of callus in subcultures of Picea wilsonii.Acta Botan Sin,1994,36(12):934-939(in Chinese).杨映根,桂耀林,唐巍,等.青杄愈伤组织在继代培养中的分化能力及染色体稳定性研究.植物学报,1994,36(12):934-939.
[18]Salaj T,BlehováA,Salaj J.Embryogenic suspension cultures of Pinus nigra Arn:Growth parameters and maturation ability.Acta Physiol Plant,2007,29(3):225-231.
[19]Yu B,Liu JM,Liu XR,et al.An efficient system of embryogenic cell suspension cultures and plant regeneration in Spathiphyllum cannifolium.Acta Horticultu Sin,2015,42(4):721-738(in Chinese).于波,刘金梅,刘晓荣,等.白鹤芋胚性细胞悬浮培养和高效植株再生体系的建立.园艺学报,2015,42(4):721-738.
[20]Mukherjee S,Ghosh B,Jha S.Establishment of forskolin yielding transfomed cell suspension cultures of Coleus forskohlii as controlledby different factors.J Biotechnol,2000,76(1):73-81.
[21]Chen YQ,Zhu WH,Wu YQ,et al.Effects of fungal elicitor on taxol prodction in suspension cells of Taxus yunnanensis.Chin J Biotech,1999,15(4):522-524(in Chinese).陈永勤,朱蔚华,吴蕴祺,等.几种真菌诱导子对云南红豆杉细胞产生紫杉醇的影响.生物工程学报,1999,15(4):522-524.
[22]Zhang CP,Li C,Yuan YJ.Effects of fungal elicitor on secondary metabolism of cell suspension culture of Taxus chinensis var.mairei.J Chem Ind Eng(China),2002,53(5):498-502(in Chinese).张长平,李春,元英进.真菌诱导子对悬浮培养南方红豆杉细胞次生代谢的影响.化工学报,2002,53(5):498-502.
[23]Sun Z,Yuan LH,Wu PM.Effects of elicitors on saffron pigment production in cell suspension cultures of Crocus sativus L..Chin J Bioprs Eng,2013,11(3):18-23(in Chinese).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.生物加工过程,2013,11(3):18-23.
[24]Dong YL,Pan XW.Effects of copper ion induction elicitation on camptothecin biosynthesis in cell suspension culture of campototheca acuminata decaisne.J Anhui Agric Sci,2010,38(23):12457-12459(in Chinese).董妍玲,潘学武.Cu2+诱导刺激对喜树悬浮培养细胞喜树碱生物合成的影响.安徽农业科学,2010,38(23):12457-12459.
[25]Almagro L,García-Pérez P,Belchí-Navarro S,et al.New strategies for the use of Linum usitatissimum cell factories for the production of bioactive compounds.Plant Physiol Biochem,2015,99:73-78.
[26]Zhao J,Zhu W H,Hu Q.Enhanced catharanthine production in Catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors.Enzyme Microb Technol,2001,28(7/8):673-681.