六盘水小黄姜脱毒快繁技术及遗传变异研究
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摘要
以贵州省六盘水市六枝特区折溪乡小黄姜姜芽为外植体,通过外植体灭菌实验确定了一套高效的姜芽外植体灭菌方法,即剥去姜芽外层组织后,用75%酒精处理30 s,0.1%升汞处理3 min。该方法可在保证存活率为100%时,达到99%的灭菌率。本研究采用正交方法检测不同浓度生长素萘乙酸(1-naphthylacetic acid,NAA)和细胞分裂素6-苄氨基腺嘌呤(6-benzylaminopurine,6-BA)对六盘水小黄姜组培苗生长的影响。数据结果表明,在接种3个月内,正交组平均长出丛生芽1.7个,不定根3.1条。当培养基中激素含量为1.0 mg/L NAA和4.0 mg/L 6-BA时,平均长出丛生芽4.1个,不定根12条。说明该激素浓度组合是促进组培苗丛生芽和不定根生长及分化的最优组合。烟草花叶病毒(tobacco mosaic virus,TMV)酶联检测显示脱毒小黄姜组培苗TMV浓度平均值为0.299 5 ng/m L(未处理小黄姜TMV浓度为3.317 4 ng/m L),说明结合茎尖脱毒与热处理脱毒可以有效除去小黄姜体内的TMV。采用ISSR分子标记技术对小黄姜脱毒苗进行遗传变异分析,从100条ISSR引物中筛选出5条稳定的多态性引物,不同继代小黄姜脱毒苗遗传变异检测表明继代培养8次后的植株与母株的遗传特性均保持一致。
A set of efficient ginger bud explant sterilization methods were identified through explant sterilization experiments using Liupanshui small yellow ginger as material, namely stripped bud outer organization and treated with 75% alcohol 30 s, 0.1% Hg Cl23 min. This method could ensure 100% survival rate and reach 99% of the sterilization rate. In this study, the effects of different concentrations of NAA and 6-BA on the growth of ginger tissue culture seedlings were detected using orthogonal method. The data showed that within 3 months after inoculation, the orthogonal group grow cluster buds were at the average of 1.7 and roots 3.1. When the medium hormone content was 0.1 mg/L NAA and 4 mg/L 6-BA, the differentiation and growth of the multiple shoot and adventitious of ginger had a significant advantage, the average of multiple shoot and adventitious were at the average of 4.1 and 12, respectively. Thus, we inferred that this concentration of the hormone could better promotethe differentiation and growth of mu ltiple shoot and adventitious roots on tissue culture seedlings. This study analyzed the influence of different hormone concentration on the differentiation and growth of ginger buds and explored the optimal growth condition of ginger buds explant culture. ELISA detection showed that the average TMV concentration of virus-free seedling was 0.299 5 ng/m L(The untreated seedling TMV concentration was3.317 4 ng/m L), The results showed that combined with virus free of shoot tip and heat treatment could effectively remove the TMV from the small yellow ginger. ISSR molecular markers were used to analyze the genetic variation of virus-free seedlings of small yellow ginger; five stable polymorphism primers were screened from 100 ISSR primers, genetic variation of different subculture seedlings showed that the genetic characteristic soft and plant were consistent after 8 subcultures.
A set of efficient ginger bud explant sterilization methods were identified through explant sterilization experiments using Liupanshui small yellow ginger as material, namely stripped bud outer organization and treated with 75% alcohol 30 s, 0.1% Hg Cl23 min. This method could ensure 100% survival rate and reach 99% of the sterilization rate. In this study, the effects of different concentrations of NAA and 6-BA on the growth of ginger tissue culture seedlings were detected using orthogonal method. The data showed that within 3 months after inoculation, the orthogonal group grow cluster buds were at the average of 1.7 and roots 3.1. When the medium hormone content was 0.1 mg/L NAA and 4 mg/L 6-BA, the differentiation and growth of the multiple shoot and adventitious of ginger had a significant advantage, the average of multiple shoot and adventitious were at the average of 4.1 and 12, respectively. Thus, we inferred that this concentration of the hormone could better promotethe differentiation and growth of mu ltiple shoot and adventitious roots on tissue culture seedlings. This study analyzed the influence of different hormone concentration on the differentiation and growth of ginger buds and explored the optimal growth condition of ginger buds explant culture. ELISA detection showed that the average TMV concentration of virus-free seedling was 0.299 5 ng/m L(The untreated seedling TMV concentration was3.317 4 ng/m L), The results showed that combined with virus free of shoot tip and heat treatment could effectively remove the TMV from the small yellow ginger. ISSR molecular markers were used to analyze the genetic variation of virus-free seedlings of small yellow ginger; five stable polymorphism primers were screened from 100 ISSR primers, genetic variation of different subculture seedlings showed that the genetic characteristic soft and plant were consistent after 8 subcultures.
引文
Altahtawy R.,Elbastawesy A.,Monem M.,Zekry Z.,Almehdar H.and Elmerzabani M.,2011,Antioxidant activity of the volatile oils of Zingiber officinale ginger,Spatula DD-Peer Reviewed Journal on Complementary Medicine and Drug Discovery,1(1):1
Bake R.,1972,Thermotherapy of planting material,Phytopathd,52:1244-1255
Bao L.,1993,Progress of ginger abroad,Xinjiang Zhongyiyao(Xinjiang Journal of Traditional Chinese Medicine),(2):52-53(包力,1993,生姜国外研究进展,新疆中医药,(2):52-53)
Chen C.Y.,Liu T.Z.,Liu Y.W.,Tseng W.C.,Liu R.H.,Lu F.J.,Lin Y.S.,Kuo S.H.,and Chen C.H.,2007,6-Shogaol(alkanone from ginger)induces apoptotic cell death of human hepatoma p53 mutant mahlavu subline via an oxidative stress-mediated caspase-dependent mechanism,Journal of Agricultural and Food Chemistry,55(3):948-954Deng N.F.,and Pan B.M,2009,Research progress on tissue culture of ginger,Anhui Nongye Kexue(Journal of Anhui Agricultural Science),37(26):12406-12410(邓年方,潘百明,2009,姜的组织培养研究进展,安徽农业科学,37(26):12406-12410)
Fan G.Q.,Wang J.Y.,Zhang P.,Ren Q.S.,Shi X.J.,and Zhao M.G.,1999,Identification pathogen of ginger and study on ginger Virus-free Rapid Propagation,In:Organizing committee of International Workshop on processing technology and industrialization of fruits and vegetables(ed.),Proceedings of the International Symposium on Fruit/Vegetable Processing Technology and Industrialization,China Science and Technology Press,Yantai,China,pp.119-136(范国强,王教义,张平,任清盛,史秀娟,赵明国,1999,姜病毒病病原鉴定及脱毒组培技术研究,见:果蔬加工技术与产业化国际研讨会组委会,编著,果蔬加工技术与产业化国际研讨会论文集,中国科学技术出版社,中国,烟台,pp.119-136)
Feng X.H.,Li J.,and Luo X.G.,2010,Progress in plant tissue culture somaclonal variation,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),26(14):70-73(丰先红,李健,罗孝贵,2010,植物组织培养中体细胞无性系变异研究,中国农学通报,26(14):70-73)
Gao S.L.,and Bian Y.Y.,1999,Tissue culture of ginger in virus-free,rapid propagation and high-yielding cultivation,Zhongguo Shucai(China Vegetables),1(3):40-41(高山林,卞云云,1999,生姜组织培养脱病毒,快繁和高产栽培,中国蔬菜,1(3):40-41)
Guo Z.X.,Miao S.,and Zhang Y.S.,2002,De-virus and rapid propagation of Zingiber officinale cv.xiao huang ginger from Luoping,Yunnan,by the tissue culture of stem apices,Yunnan Daxue Xuebao(Journal of Yunnan University(Natural Sciences Edition)),24(6):473-474(郭忠新,缪森,张云孙,2002,云南罗平小黄姜的茎尖脱毒组培快繁,云南大学学报(自然科学版),24(6):4731474)
Huang H.F.,and Zhang Y.L.,2016,Process of candied ginger in Luoping,Shiping Yanjiu Yu Kaifa(Food Research and Development),37(8):120-123(黄慧福,张玉丽,2016,罗平小黄姜姜脯的工艺研究,食品研究与开发,37(8):120-123)
Huang J.H.,and Chen S.R.,1991,In vitro Conservation Technique of Germplasm in Ginger,Xinan Nongye Xuebao(Journal of Southwest Agricultural University),13(3):310-313(黄菊辉,陈世儒,1991,生姜种质资源离体繁殖和保存,西南农业大学学报,13(3):310-313)
Jin X.J.,Yang L.,and Liu Z.L.,2004,Diarylheptanoids from the rhizomes of Zingiber officinale,Phytochemistry,65:1137-1143
Jin Y.,Zhang W.J.,Fu D.X.,and Lu B.R.,2003,Sampling strategywithin a wild soybean population based on its genetic variation detected by ISSR markers,Acta Agriculturae Nucleatae Sinica,45(8):995-1002
Jolad S.D.,Lantz R.C.,Solyom A.M.,Chen G.J.,Bates R.B.,and Timmermann B.N.,2004,Fresh organically grown ginger(Zingiber officinale):Composition and effects on LPSinduced PGE2 production,Phytochemistry,65:1937-1954
Lei K.R.,2006,Study on synchronous culture of proliferation and rooting in ginger tissue culture,Zhonggu Zhongye(China Seed Industry),(5):33-34(雷开荣,2006,生姜组织培养中增殖与生根同步培养技术研究,中国种业,(5):33-34)
Lin B.Y.,Wei Z.Z.,and Chen Y.H.,2002,Tissue culture and rapid propagation of Zingiber officinale,Yaredai Zhiwu Kexue(Subtropical Plant Science),31(4):13-16(林碧英,魏郑珍,陈燕华,2002,生姜茎尖组织培养和快速繁殖研究,亚热带植物科学,31(4):13-16)
Liu B.,Miao J.,and Wu X.,2011,Research progress of ginger,Heilongjiang Nongye Kexue(Heilongjiang Agricultural Science),(5):135-138(刘波,缪军,吴雄,2011,生姜研究进展,黑龙江农业科学,(5):135-138)
Li W.G.,Chang T.J.,and Gong H.M.,2008,Methylationsensitive amplified polymorphism and its application to plant genetics,Shengwu Jishu(Biotechnology),18(1):83-87(李卫国,常天俊,龚红梅,2008,MSAP技术及其在植物遗传学研究中的应用,生物技术,18(1):83-87)
Li X.L.,Chen J.,and Wang G.,2008,spatial autocorrelation analysis of ISSR genetic variation of Reaumuria soongorica population in north west of China,Zhongguo Shamo(Journal of Desert Research),28(3):468-472(李秀玲,陈健,王刚,2008,西北地区红砂种群ISSR遗传变异的空间自相关分析,中国沙漠,28(3):468-472)
Luo T.K.,and Zhang X.L.,2005,Study on the technology of detoxification and rapid propagation of Taiwan ginger,Wenzhou Nongye Keji(Wenzhou Agricultural Science and Technology),4:22-25(罗天宽,张小玲,2005,台湾肉姜脱菌脱毒快繁技术研究,温州农业科技,4:22-25)
Nalbantsoy,A.,Tamis I.D.A.,Akgün H.,Yalcin T.魻.,Gürhan I.D.,and Karaboz I.,2008,Antimicrobial and cytotoxic activities of Zingiber officinalis extracts,Journal of Pharmacy&Pharmaceutical Sciences,33(2):78-85
Nie C.P.,Yang P.P.,Ma Z.X.,and Li Y.Y.,2016,Effects ofγray irradiation with different dosages on strawberry seeds and their genetic diversity revealed by ISSR,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(12):1048-1054(聂传朋,杨盼盼,马宗新,李焰焰,2016,基于ISSR揭示草莓种子不同剂量γ辐射效果及遗传多样性,分子植物育种,14(12):1048-1054)
Qian D.S.,and Liu Z.S.,1992,Pharmacologic studies of antimotion sickness actions of ginger,Chinese Journal of Integrated Traditional&Western Medicine,12(2):95
Qi L.,Zhu H.L.,Chen J.X.,and Bai C.Y.,2013,Virus elimination and rapid propagation of small yellow ginger(Zingiber officinale Rosc)in Hainan,Redai Nongye Xuebao(Chinese Journal of Tropical Agriculture),33(9):37-40(齐兰,朱红林,陈健晓,白翠云,2013,海南小黄姜脱毒快繁技术研究,热带农业科学,33(9):37-40)
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Yu H.L.,Wang F.G.,and Li S.Y.,2014,Assessment of genetic stability of leaves in vitro regenerated from oriental lily by ISSR,Yaredai Nongye Yanjiu(Subtropical Agriculture Research),10(1):35-40(于海玲,王发国,李仕裕,2014,东方百合离体再生前后叶片遗传稳定性的ISSR检测,亚热带农业研究,10(1):35-40)
Zhang Y.,Tian X.M.,Zhou Z.C.,Liu W.B.,Chu D.Y.,Qin G.F.,and Jin G.Q.,2010,Genetic variation of the second-generation breeding population of masson pine based on major growth traits and ISSR,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),8(3):501-510(张一,谭小梅,周志春,刘伟宏,储德裕,秦国峰,金国庆,2010,马尾松二代育种亲本主要生长性状和ISSR遗传变异,分子植物育种,8(3):501-510)
Bake R.,1972,Thermotherapy of planting material,Phytopathd,52:1244-1255
Bao L.,1993,Progress of ginger abroad,Xinjiang Zhongyiyao(Xinjiang Journal of Traditional Chinese Medicine),(2):52-53(包力,1993,生姜国外研究进展,新疆中医药,(2):52-53)
Chen C.Y.,Liu T.Z.,Liu Y.W.,Tseng W.C.,Liu R.H.,Lu F.J.,Lin Y.S.,Kuo S.H.,and Chen C.H.,2007,6-Shogaol(alkanone from ginger)induces apoptotic cell death of human hepatoma p53 mutant mahlavu subline via an oxidative stress-mediated caspase-dependent mechanism,Journal of Agricultural and Food Chemistry,55(3):948-954Deng N.F.,and Pan B.M,2009,Research progress on tissue culture of ginger,Anhui Nongye Kexue(Journal of Anhui Agricultural Science),37(26):12406-12410(邓年方,潘百明,2009,姜的组织培养研究进展,安徽农业科学,37(26):12406-12410)
Fan G.Q.,Wang J.Y.,Zhang P.,Ren Q.S.,Shi X.J.,and Zhao M.G.,1999,Identification pathogen of ginger and study on ginger Virus-free Rapid Propagation,In:Organizing committee of International Workshop on processing technology and industrialization of fruits and vegetables(ed.),Proceedings of the International Symposium on Fruit/Vegetable Processing Technology and Industrialization,China Science and Technology Press,Yantai,China,pp.119-136(范国强,王教义,张平,任清盛,史秀娟,赵明国,1999,姜病毒病病原鉴定及脱毒组培技术研究,见:果蔬加工技术与产业化国际研讨会组委会,编著,果蔬加工技术与产业化国际研讨会论文集,中国科学技术出版社,中国,烟台,pp.119-136)
Feng X.H.,Li J.,and Luo X.G.,2010,Progress in plant tissue culture somaclonal variation,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),26(14):70-73(丰先红,李健,罗孝贵,2010,植物组织培养中体细胞无性系变异研究,中国农学通报,26(14):70-73)
Gao S.L.,and Bian Y.Y.,1999,Tissue culture of ginger in virus-free,rapid propagation and high-yielding cultivation,Zhongguo Shucai(China Vegetables),1(3):40-41(高山林,卞云云,1999,生姜组织培养脱病毒,快繁和高产栽培,中国蔬菜,1(3):40-41)
Guo Z.X.,Miao S.,and Zhang Y.S.,2002,De-virus and rapid propagation of Zingiber officinale cv.xiao huang ginger from Luoping,Yunnan,by the tissue culture of stem apices,Yunnan Daxue Xuebao(Journal of Yunnan University(Natural Sciences Edition)),24(6):473-474(郭忠新,缪森,张云孙,2002,云南罗平小黄姜的茎尖脱毒组培快繁,云南大学学报(自然科学版),24(6):4731474)
Huang H.F.,and Zhang Y.L.,2016,Process of candied ginger in Luoping,Shiping Yanjiu Yu Kaifa(Food Research and Development),37(8):120-123(黄慧福,张玉丽,2016,罗平小黄姜姜脯的工艺研究,食品研究与开发,37(8):120-123)
Huang J.H.,and Chen S.R.,1991,In vitro Conservation Technique of Germplasm in Ginger,Xinan Nongye Xuebao(Journal of Southwest Agricultural University),13(3):310-313(黄菊辉,陈世儒,1991,生姜种质资源离体繁殖和保存,西南农业大学学报,13(3):310-313)
Jin X.J.,Yang L.,and Liu Z.L.,2004,Diarylheptanoids from the rhizomes of Zingiber officinale,Phytochemistry,65:1137-1143
Jin Y.,Zhang W.J.,Fu D.X.,and Lu B.R.,2003,Sampling strategywithin a wild soybean population based on its genetic variation detected by ISSR markers,Acta Agriculturae Nucleatae Sinica,45(8):995-1002
Jolad S.D.,Lantz R.C.,Solyom A.M.,Chen G.J.,Bates R.B.,and Timmermann B.N.,2004,Fresh organically grown ginger(Zingiber officinale):Composition and effects on LPSinduced PGE2 production,Phytochemistry,65:1937-1954
Lei K.R.,2006,Study on synchronous culture of proliferation and rooting in ginger tissue culture,Zhonggu Zhongye(China Seed Industry),(5):33-34(雷开荣,2006,生姜组织培养中增殖与生根同步培养技术研究,中国种业,(5):33-34)
Lin B.Y.,Wei Z.Z.,and Chen Y.H.,2002,Tissue culture and rapid propagation of Zingiber officinale,Yaredai Zhiwu Kexue(Subtropical Plant Science),31(4):13-16(林碧英,魏郑珍,陈燕华,2002,生姜茎尖组织培养和快速繁殖研究,亚热带植物科学,31(4):13-16)
Liu B.,Miao J.,and Wu X.,2011,Research progress of ginger,Heilongjiang Nongye Kexue(Heilongjiang Agricultural Science),(5):135-138(刘波,缪军,吴雄,2011,生姜研究进展,黑龙江农业科学,(5):135-138)
Li W.G.,Chang T.J.,and Gong H.M.,2008,Methylationsensitive amplified polymorphism and its application to plant genetics,Shengwu Jishu(Biotechnology),18(1):83-87(李卫国,常天俊,龚红梅,2008,MSAP技术及其在植物遗传学研究中的应用,生物技术,18(1):83-87)
Li X.L.,Chen J.,and Wang G.,2008,spatial autocorrelation analysis of ISSR genetic variation of Reaumuria soongorica population in north west of China,Zhongguo Shamo(Journal of Desert Research),28(3):468-472(李秀玲,陈健,王刚,2008,西北地区红砂种群ISSR遗传变异的空间自相关分析,中国沙漠,28(3):468-472)
Luo T.K.,and Zhang X.L.,2005,Study on the technology of detoxification and rapid propagation of Taiwan ginger,Wenzhou Nongye Keji(Wenzhou Agricultural Science and Technology),4:22-25(罗天宽,张小玲,2005,台湾肉姜脱菌脱毒快繁技术研究,温州农业科技,4:22-25)
Nalbantsoy,A.,Tamis I.D.A.,Akgün H.,Yalcin T.魻.,Gürhan I.D.,and Karaboz I.,2008,Antimicrobial and cytotoxic activities of Zingiber officinalis extracts,Journal of Pharmacy&Pharmaceutical Sciences,33(2):78-85
Nie C.P.,Yang P.P.,Ma Z.X.,and Li Y.Y.,2016,Effects ofγray irradiation with different dosages on strawberry seeds and their genetic diversity revealed by ISSR,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(12):1048-1054(聂传朋,杨盼盼,马宗新,李焰焰,2016,基于ISSR揭示草莓种子不同剂量γ辐射效果及遗传多样性,分子植物育种,14(12):1048-1054)
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