杜鹃品种‘江南春早’叶片离体再生体系的建立
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摘要
以杜鹃品种‘江南春早’无菌苗叶片为外植体,研究了基本培养基类型、暗培养时间、不同叶位及不同激素组合等因素对叶片再生不定芽的影响,建立了‘江南春早’叶片再生体系。研究结果表明:WPM培养基为最适基本培养基。初始暗培养有利于叶片再生不定芽,暗培养20 d的效果最佳。‘江南春早’组培苗中、上部叶片再生能力较强,其中第3和第4叶位叶片的不定芽诱导率最高。诱导‘江南春早’组培苗叶片再生不定芽的适宜培养基为WPM+CPPU 3.0 mg/L+IAA 1.0 mg/L,不定芽诱导率和平均不定芽数分别达到70.0%和7.4个。本研究初步建立了‘江南春早’叶片再生体系,为‘江南春早’繁育良种试管苗以及后续遗传转化体系的建立提供了科学依据。
Aseptic seedling leaves of Rhododendron cultivar 'Jiangnan Chunzao' were used as explants, the effects of basic culture medium type, dark culture time, different leaf position and hormone combinations on adventitious buds regeneration were studied, and the regeneration system of 'Jiangnan Chunzao' leaves was established. The results showed that WPM medium was the optimal basic culture medium. The initial dark culture was beneficial to the regeneration of adventitious buds and the effect of dark culture for 20 days was the best. The regeneration ability of leaves at upper and middle parts of a plantlet was stronger, and the adventitious buds induction rate of leaves at the 3 th and 4 th positions was the highest. The optimum medium for adventitious buds regeneration of in vitro leaves was WPM+CPPU 3.0 mg/L+IAA 1.0 mg/L, and the induction rate was 70.0% as well as the average number of adventitious buds was 7.4. In this study, the leaf regeneration system of 'Jiangnan Chunzao' was preliminarily established, which would provide a scientific basis for the breeding of test-tube plantlet of 'Jiangnan Chunzao' and the establishment of subsequent genetic transformation system.
Aseptic seedling leaves of Rhododendron cultivar 'Jiangnan Chunzao' were used as explants, the effects of basic culture medium type, dark culture time, different leaf position and hormone combinations on adventitious buds regeneration were studied, and the regeneration system of 'Jiangnan Chunzao' leaves was established. The results showed that WPM medium was the optimal basic culture medium. The initial dark culture was beneficial to the regeneration of adventitious buds and the effect of dark culture for 20 days was the best. The regeneration ability of leaves at upper and middle parts of a plantlet was stronger, and the adventitious buds induction rate of leaves at the 3 th and 4 th positions was the highest. The optimum medium for adventitious buds regeneration of in vitro leaves was WPM+CPPU 3.0 mg/L+IAA 1.0 mg/L, and the induction rate was 70.0% as well as the average number of adventitious buds was 7.4. In this study, the leaf regeneration system of 'Jiangnan Chunzao' was preliminarily established, which would provide a scientific basis for the breeding of test-tube plantlet of 'Jiangnan Chunzao' and the establishment of subsequent genetic transformation system.
引文
Ai P.F.,and Luo Z.R.,2002,Effect of CPPU on propagation of persimmon in vitro,Guoshu Xuebao(Journal of Fruit Science),19(4):272-274(艾鹏飞,罗正荣,2002,CPPU在柿试管繁殖中的应用,果树学报,19(4):272-274)
Cai G.J.,Zhu H.B.,Chen X.N.,Wei X.L.,and Liu H.Y.,2003,Study on in vitro fast-propagation and industrialization breeding techniques of triploid chinese white poplar,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),23(12):2188-2191(蔡国军,朱红斌,陈晓妮,魏晓兰,刘鸿源,2003,三倍体毛白杨组培快繁和工厂化育苗技术研究,西北植物学报,23(12):2188-2191)
Cai X.,and Kang X.Y.,2011,In vitro tetraploid induction from leaf explants of Populus pseudo-simonii Kitag,Plant Cell Rep.,30(9):1771-1778
Li W.J.,Cao H.N.,Zong C.W.,Jin C.,and Liu M.,2012,Establishment of regeneration system of'Pingguoli'pear(Pyrus breschneideri)leaves in vitro,Guoshu Xuebao(Journal of Fruit Science),29(5):800-803(李文剑,曹后男,宗成文,金灿,刘淼,2012,‘苹果梨’离体叶片再生体系的建立,果树学报,29(5):800-803)
Lin F.,Li J.Y.,Huang D.M.,Xu Y.,Li Y.J.,Li Y.X.,Ding Z.L.,and Tang F.L.,2015,Study of tissue culture and plant regeneration of Aquilaria sinensis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(6):1296-1299(林妃,李敬阳,黄东梅,许奕,李羽佳,李艳霞,丁哲利,唐粉玲,2015,白木香组织培养技术及植株再生的研究,基因组学与应用生物学,34(6):1296-1299)
Liu X.Q.,Su J.L.,Li C.,He L.S.,and Chen L.,2011,A new alpine rhododendron cultivar'Fuli Jinling',Yuanyi Xuebao(Acta Horticulturae Sinica),38(11):2237-2238(刘晓青,苏家乐,李畅,何丽斯,陈璐,2011,高山杜鹃新品种‘富丽金陵’,园艺学报,38(11):2237-2238)
Liu X.Q.,Su J.L.,Li C.,He L.S.,and Chen L.,2015,A new Rhododendron cultivar'Jiangnan Chunzao',Yuanyi Xuebao(Acta Horticulturae Sinica),42(5):1015-1016(刘晓青,苏家乐,李畅,何丽斯,陈璐,2015,杜鹃花新品种‘江南春早’,园艺学报,42(5):1015-1016)
Lv X.L.,Wei X.Y.,Yin L.J.,Huang F.,and Zhang C.Y.,2014,In vitro leave culture of broad leaf evergreen rhododendron and its regeneration of adventitious buds,Nanjing Lingye Daxue Xuebao(Journal of Nanjing Forestry University),38(2):165-168(吕秀立,魏翔莺,尹丽娟,黄芳,张春英,2014,大叶常绿杜鹃叶片离体培养及不定芽再生,南京林业大学学报,38(2):165-168)
Magyar-Tabori K.,Dobranszki J.,Silva J.A.T.D.,Bulley S.M.,and Hudak I.,2010,The role of cytokinins in shoot organogenesis in apple,Plant Cell Tiss,Organ.Cult.,101(3):251-267
Miao Y.M.,Wang Y.Q.,Zhuang P.,and Jian X.,2006,Study on technique tissue culture in Rhododendron anne franch,Shengwuxue Zazhi(Journal of Biology),23(6):29-32(苗永美,王永清,庄平,简兴,2006,桃叶杜鹃组织培养技术研究,生物学杂志,23(6):29-32)
Pavingerova D.,2009,The influence of thidiazuron on shoot regeneration from leaf explants of fifteen cultivars of Rhododendron,Biologia Plantarum,53(4):797-799
Reynolds A.G.,Wardle D.A.,Zurowski C.,and Looney N.E.,1992,Phenylureas CPPU and thidiazuron affect yield components,fruit composition,and storage potential of four seedless grape selections,J.Amer.Soc.Hort.Sci.,117(1):85-89
Shu Y.J.,Lu Y.,Tang Y.C.,Hu N.B.,and Zhou Y.L.,2016,Establishment of in vitro regeneration system of Mentha(Mentha haplocalyx)leaves,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(12):3483-3488(舒英杰,陆艳,唐玉超,胡能兵,周玉丽,2016,薄荷叶片离体再生体系的建立,分子植物育种,14(12):3483-3488)
Su J.L.,Liu X.Q.,Li C.,He L.S.,and Chen L.,2012,A new Rhododendron cultivar'Yanzhi Mi',Yuanyi Xuebao(Acta Horticulturae Sinica),39(12):2555-2556(苏家乐,刘晓青,李畅,何丽斯,陈璐,2012,杜鹃花新品种‘胭脂蜜’,园艺学报,39(11):2555-2556)
Wang F.,Gao Z.H.,Zhang Z.,Qiao Y.S.,Wang M.,Li G.P.,and Mi L.,2010,Stablishment of regeneration system of strawberry,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),30(5):1045-1049(王翡,高志红,章镇,乔玉山,王萌,李国平,糜林,2010,草莓高效离体叶片再生体系的建立,西北植物学报,30(5):1045-1049)
Wang G.L.,Fang H.J.,and Na J.,1997,Application of potent cytokinin-thidiazuron(TDZ)to plant,Zhiwuxue Tongbao(C-hinese Bulletin of Botany),14(3):47-53(王关林,方宏筠,那杰,1997,高活性细胞激动素TDZ在植物组织培养中的应用,植物学通报,14(3):47-53)
Wang L.B.,Zhang C.H.,Wang X.M.,Wu W.L.,and Li W.L.,2012,Analysis of influence factors of adventitious bud induction from in vitro leaves of blackberry cultivar'Arapaho'and establishment of regeneration system,Zhiwu Ziyuan Yu Huanjing Xuebao(Journal of Plant Resources and Environment),21(1):47-51(王鲁北,张春红,王小敏,吴文龙,李维林,2012,黑莓品种‘Arapaho’离体叶片不定芽诱导影响因素分析及再生体系建立,植物资源与环境学报,21(1):47-51)
Wedzony M.,Szechynska-Hebda M.,Zur I.,Dubas E.,and Krzewska M.,2014,Tissue culture and regeneration:a prerequisite for alien gene transfer,Alien Gene Transfer in Crop Plants,6(1):43-75
Zaytseva Y.G.,Poluboyarova T.V.,and Novikova T.I.,2016,Effects of thidiazuron on in vitro morphogenic response of Rhododendron sichotense Pojark.and Rhododendron catawbiense cv.Grandiflorum leaf explants,In Vitro Cell Dev.B-iol.Plant,52(1):56-63
Cai G.J.,Zhu H.B.,Chen X.N.,Wei X.L.,and Liu H.Y.,2003,Study on in vitro fast-propagation and industrialization breeding techniques of triploid chinese white poplar,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),23(12):2188-2191(蔡国军,朱红斌,陈晓妮,魏晓兰,刘鸿源,2003,三倍体毛白杨组培快繁和工厂化育苗技术研究,西北植物学报,23(12):2188-2191)
Cai X.,and Kang X.Y.,2011,In vitro tetraploid induction from leaf explants of Populus pseudo-simonii Kitag,Plant Cell Rep.,30(9):1771-1778
Li W.J.,Cao H.N.,Zong C.W.,Jin C.,and Liu M.,2012,Establishment of regeneration system of'Pingguoli'pear(Pyrus breschneideri)leaves in vitro,Guoshu Xuebao(Journal of Fruit Science),29(5):800-803(李文剑,曹后男,宗成文,金灿,刘淼,2012,‘苹果梨’离体叶片再生体系的建立,果树学报,29(5):800-803)
Lin F.,Li J.Y.,Huang D.M.,Xu Y.,Li Y.J.,Li Y.X.,Ding Z.L.,and Tang F.L.,2015,Study of tissue culture and plant regeneration of Aquilaria sinensis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(6):1296-1299(林妃,李敬阳,黄东梅,许奕,李羽佳,李艳霞,丁哲利,唐粉玲,2015,白木香组织培养技术及植株再生的研究,基因组学与应用生物学,34(6):1296-1299)
Liu X.Q.,Su J.L.,Li C.,He L.S.,and Chen L.,2011,A new alpine rhododendron cultivar'Fuli Jinling',Yuanyi Xuebao(Acta Horticulturae Sinica),38(11):2237-2238(刘晓青,苏家乐,李畅,何丽斯,陈璐,2011,高山杜鹃新品种‘富丽金陵’,园艺学报,38(11):2237-2238)
Liu X.Q.,Su J.L.,Li C.,He L.S.,and Chen L.,2015,A new Rhododendron cultivar'Jiangnan Chunzao',Yuanyi Xuebao(Acta Horticulturae Sinica),42(5):1015-1016(刘晓青,苏家乐,李畅,何丽斯,陈璐,2015,杜鹃花新品种‘江南春早’,园艺学报,42(5):1015-1016)
Lv X.L.,Wei X.Y.,Yin L.J.,Huang F.,and Zhang C.Y.,2014,In vitro leave culture of broad leaf evergreen rhododendron and its regeneration of adventitious buds,Nanjing Lingye Daxue Xuebao(Journal of Nanjing Forestry University),38(2):165-168(吕秀立,魏翔莺,尹丽娟,黄芳,张春英,2014,大叶常绿杜鹃叶片离体培养及不定芽再生,南京林业大学学报,38(2):165-168)
Magyar-Tabori K.,Dobranszki J.,Silva J.A.T.D.,Bulley S.M.,and Hudak I.,2010,The role of cytokinins in shoot organogenesis in apple,Plant Cell Tiss,Organ.Cult.,101(3):251-267
Miao Y.M.,Wang Y.Q.,Zhuang P.,and Jian X.,2006,Study on technique tissue culture in Rhododendron anne franch,Shengwuxue Zazhi(Journal of Biology),23(6):29-32(苗永美,王永清,庄平,简兴,2006,桃叶杜鹃组织培养技术研究,生物学杂志,23(6):29-32)
Pavingerova D.,2009,The influence of thidiazuron on shoot regeneration from leaf explants of fifteen cultivars of Rhododendron,Biologia Plantarum,53(4):797-799
Reynolds A.G.,Wardle D.A.,Zurowski C.,and Looney N.E.,1992,Phenylureas CPPU and thidiazuron affect yield components,fruit composition,and storage potential of four seedless grape selections,J.Amer.Soc.Hort.Sci.,117(1):85-89
Shu Y.J.,Lu Y.,Tang Y.C.,Hu N.B.,and Zhou Y.L.,2016,Establishment of in vitro regeneration system of Mentha(Mentha haplocalyx)leaves,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(12):3483-3488(舒英杰,陆艳,唐玉超,胡能兵,周玉丽,2016,薄荷叶片离体再生体系的建立,分子植物育种,14(12):3483-3488)
Su J.L.,Liu X.Q.,Li C.,He L.S.,and Chen L.,2012,A new Rhododendron cultivar'Yanzhi Mi',Yuanyi Xuebao(Acta Horticulturae Sinica),39(12):2555-2556(苏家乐,刘晓青,李畅,何丽斯,陈璐,2012,杜鹃花新品种‘胭脂蜜’,园艺学报,39(11):2555-2556)
Wang F.,Gao Z.H.,Zhang Z.,Qiao Y.S.,Wang M.,Li G.P.,and Mi L.,2010,Stablishment of regeneration system of strawberry,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),30(5):1045-1049(王翡,高志红,章镇,乔玉山,王萌,李国平,糜林,2010,草莓高效离体叶片再生体系的建立,西北植物学报,30(5):1045-1049)
Wang G.L.,Fang H.J.,and Na J.,1997,Application of potent cytokinin-thidiazuron(TDZ)to plant,Zhiwuxue Tongbao(C-hinese Bulletin of Botany),14(3):47-53(王关林,方宏筠,那杰,1997,高活性细胞激动素TDZ在植物组织培养中的应用,植物学通报,14(3):47-53)
Wang L.B.,Zhang C.H.,Wang X.M.,Wu W.L.,and Li W.L.,2012,Analysis of influence factors of adventitious bud induction from in vitro leaves of blackberry cultivar'Arapaho'and establishment of regeneration system,Zhiwu Ziyuan Yu Huanjing Xuebao(Journal of Plant Resources and Environment),21(1):47-51(王鲁北,张春红,王小敏,吴文龙,李维林,2012,黑莓品种‘Arapaho’离体叶片不定芽诱导影响因素分析及再生体系建立,植物资源与环境学报,21(1):47-51)
Wedzony M.,Szechynska-Hebda M.,Zur I.,Dubas E.,and Krzewska M.,2014,Tissue culture and regeneration:a prerequisite for alien gene transfer,Alien Gene Transfer in Crop Plants,6(1):43-75
Zaytseva Y.G.,Poluboyarova T.V.,and Novikova T.I.,2016,Effects of thidiazuron on in vitro morphogenic response of Rhododendron sichotense Pojark.and Rhododendron catawbiense cv.Grandiflorum leaf explants,In Vitro Cell Dev.B-iol.Plant,52(1):56-63
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