不同培养基组分对带叶兜兰离体培养效果的影响
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摘要
【目的】探讨适宜带叶兜兰(Paphiopedilum hirsutissimum)离体培养的培养基组分,为规模化生产带叶兜兰组培苗提供技术支持。【方法】以带叶兜兰组培苗为外植体,筛选适合其丛生芽诱导、增殖和壮苗生长的基本培养基、附加物种类和生长激素组合。【结果】初步筛选出较适宜带叶兜兰丛生芽诱导的培养基为1/2MS+3.0 mg/L BA+0.1 mg/LNAA+80.0 g/L香蕉汁+2.0 g/L活性炭,其诱导率较高,为36.1%,且丛生芽诱导出芽较多;丛生芽增殖培养基为1/2MS+0.1 mg/L TDZ+3.0 mg/L 2,4-D+80.0 g/L香蕉汁+2.0 g/L活性炭,其增殖系数为2.02,芽生长较快;壮苗生长最适宜培养基为1.0 g/L花宝1号+1.0 g/L花宝2号+MS培养基的有机、微量成分+1.0 mg/LNAA+2.0 g/L活性炭+80.0 g/L香蕉汁+1.5g/L蛋白胨,其幼苗生长较好,生物量大,生根率达75.5%。【结论】在1/2MS+3.0 mg/L6-BA+0.1 mg/LNAA+2.0 g/L活性炭培养基中添加香蕉汁较适宜带叶兜兰丛生芽诱导和增殖培养;在1/2MS+80.0g/L香蕉汁培养基中添加0.1 mg/LTDZ+3.0 mg/L 2,4-D对带叶兜兰的增殖效果较佳;花宝改良培养基适宜带叶兜兰壮苗培养,结合添加NAA和活性炭培养效果更佳,但活性炭浓度不宜过高。
【Objective】Medium components suitable for in vitro culture of Paphiopedilum hirsutissimum was developed in order to provide technical support for large-scale multiplication of P.hirsutissimum by in vitro tissue culture.【Method】Taking tissue culture seedlings of P.hirsutissimum as explants,basic culture medium,additives and hormone combination,which were suitable for bud induction,multiplication of cluster buds and seedling growuS of P.hirsutissimum,were screened.【Result】Results showed that,1/2MS+3.0 mg/L 6-BA+0.1 mg/L NAA+80.0 g/L banana juice+2.0 g/L activated carbon was optimal medium for induction of cluster buds,with induction rate of 36.1%,and more cluster buds sprouted.1/2MS+0.1 mg/L TDZ+3.0 mg/L 2,4-D+80.0 g/L banana juice+2.0 g/L activated carbon was optimal medium for multiplication of cluster buds,the multiplication multiple was 2.02,and buds grew faster on this medium.The optimal medium for strengthening P.hirsutissimum seedling was 1.0 g/L Hyponex 1 + 1.0 g/L Hyponex 2+MS(organic and micro)+ 1.0 mg/L NAA+2.0 g/L activated carbon+80.0 g/L banana juice+ 1.5 g/L peptone,seedling grew well on this medium,with large biomass and rooting rate of 75.5%.【Conclusion】Adding banana juice to medium(1/2MS+3.0 mg/L 6-BA+0.1 mg/L NAA+2.0 g/L activated carbon) is conducive to bud induction and multiplication of P.hirsutissimum,and adding 0.1 mg/L TDZ+3.0 mg/L 2,4-D to 1/2MS+80.0 g/L banana juice has good effect on multiplication.Improved Hyponex medium is suitable for strengthening P.hirsutissimum seedling,and improved Hyponex medium supplemented with NAA and activated carbon has culture effect,but amount of added activated carbon can not be too high.
【Objective】Medium components suitable for in vitro culture of Paphiopedilum hirsutissimum was developed in order to provide technical support for large-scale multiplication of P.hirsutissimum by in vitro tissue culture.【Method】Taking tissue culture seedlings of P.hirsutissimum as explants,basic culture medium,additives and hormone combination,which were suitable for bud induction,multiplication of cluster buds and seedling growuS of P.hirsutissimum,were screened.【Result】Results showed that,1/2MS+3.0 mg/L 6-BA+0.1 mg/L NAA+80.0 g/L banana juice+2.0 g/L activated carbon was optimal medium for induction of cluster buds,with induction rate of 36.1%,and more cluster buds sprouted.1/2MS+0.1 mg/L TDZ+3.0 mg/L 2,4-D+80.0 g/L banana juice+2.0 g/L activated carbon was optimal medium for multiplication of cluster buds,the multiplication multiple was 2.02,and buds grew faster on this medium.The optimal medium for strengthening P.hirsutissimum seedling was 1.0 g/L Hyponex 1 + 1.0 g/L Hyponex 2+MS(organic and micro)+ 1.0 mg/L NAA+2.0 g/L activated carbon+80.0 g/L banana juice+ 1.5 g/L peptone,seedling grew well on this medium,with large biomass and rooting rate of 75.5%.【Conclusion】Adding banana juice to medium(1/2MS+3.0 mg/L 6-BA+0.1 mg/L NAA+2.0 g/L activated carbon) is conducive to bud induction and multiplication of P.hirsutissimum,and adding 0.1 mg/L TDZ+3.0 mg/L 2,4-D to 1/2MS+80.0 g/L banana juice has good effect on multiplication.Improved Hyponex medium is suitable for strengthening P.hirsutissimum seedling,and improved Hyponex medium supplemented with NAA and activated carbon has culture effect,but amount of added activated carbon can not be too high.
引文
陈尔,王华新,陈宝玲,农凤梅,李冰,苏莉花.2014.春兰根状茎增殖的影响因素[J].广西林业科学,43(3):319-321.Chen E,Wang H X,Chen B L,Nong F M,Li B,Su L H.2014.Influence factors of Cymbidium goeringii rhizomes multiplication[J].Guangxi Forestry Science,43(3):319-321.
陈宇勒.2009.兰花繁育技术图解[M].广州:广东科技出版社.Chen Y L.2009.Orchid Breeding Technology Illustration[M].Guangzhou:Guangdong Science and Technology Publishing House.
陈之林,叶秀粦,梁承邺,段俊.2004.杏黄兜兰和硬叶兜兰的种子试管培养[J].园艺学报,31(4):540-542.Chen Z L,Ye X L,Liang C Y,Duan J.2004.Seed germination in vitro of Paphiopedilum armeniacum and P.micranthum[J].Acta Horticulturae Sinica,31(4):540-542.
崔秋华,孙永玉,李昆,周志美,段明伦.2012.不同因子对齿瓣石斛增殖系数和平均苗高的影响[J].中南林业科技大学学报,32(4):200-203.Cui Q H,Sun Y Y,Li K,Zhou Z M,Duan M L.2012.Effects of different factors on proliferation coefficient of Dendrobium devonianum and average seedling height[J].Journal of Central South University of Forestry&Technology,32(4):200-203.
丁长春,夏念和.2009.麻栗坡兜兰的无菌播种与快速繁殖[J].植物生理学通讯,45(2):1201-1202.Ding C C,Xia N H.2009.Aseptic sowing and rapid propagation of Paphiopedilum malipoense S.C.Chen&Z.H.Tsi in vitro[J].Plant Physiology Communications,45(2):1201-1202.
丁长春,虞泓,刘方媛.2004.影响杏黄兜兰种子萌发的因素[J].云南植物研究,26(6):673-677.Ding C C,Yu H.Liu F Y.2004.Factors affecting the germination of Paphiopedilum armeniacum[J].Acta Botanica Yunnanica,26(6):673-677.
李慧敏,杨冠海,李明静,闻永慧,张青华,李枝林.2013.白芨瓶内假鳞茎诱导研究[J].南方农业学报,44(10):1607-1612.Li H M,Yang G H,Li M J,Wen Y H,Zhang Q H,Li Z L.2013.Bletilla striata pseudobulb induction in hottle[J].Journal of Southern Agriculture,44(10):1607-1612.
刘仲健,刘可为,陈利君,雷嗣鹏,李利强,施晓春,黄来强.2006.濒危物种杏黄兜兰的保育生态学[J].生态学报,26(9):2791-2800.Liu Z J,Liu K W,Chen L J,Lei S P,Li L Q,Shi X C,Huang L Q.2006.Conservation ecology of endangered species Paphiopedilum armeniacum(Orchidaceae)[J].Acta Ecologica Sinica,26(9):2791-2800.
罗毅波,贾建生,王春玲.2003.初论中国兜兰属植物的保护策略及其潜在资源优势[J].生物多样性,11(6):491-498.Luo Y B,Jia J S,Wang C L.2003.Conservation strategy and potential advantages of the Chinese Paphiopedilum[J].Biodiversity Science,11(6):491-498.
秦丽凤,石贵玉.2013.外源激素对杂交兰组培快繁的效应[J].南方农业学报,44(9):1435-1438.Qin L F,Shi G Y.2013.Effects of exogenous growth regulators on rapid propagation of Cymbidium hybridum×Faberi[J].Journal of Southern Agriculture,44(9):1435-1438.
史军,程瑾,罗敦,上官法治,罗毅波.2007.利用传粉综合征预测:长瓣兜兰模拟繁殖地欺骗雌性食蚜蝇传粉[J].植物分类学报,45(4):551-560.Shi J,Cheng J,Luo D,Shangguan F Z,Luo Y B.2007.Pollination syndromes predict brood-site deceptive pollination by female hoverflies in Paphiopedilum dianthum(Orchidaceae)[J].Acta Phytotaxonomica Sinica,45(4):551-560.
孙晓颖.2014.五种野生兜兰植物菌根真菌多样性研究[D].北京:北京林业大学.Sun X Y.2014.Diversity of mycorrhizal fungi isolated from five species of the genus Paphiopedilum[D].Beijing:Beijing Forestry University.
田凡,姜运力,罗在柒,王莲辉.2014.白花兜兰种子无菌萌发及试管成苗技术研究[J].贵州林业科技,42(3):34-38.Tian F,Jiang Y L,Luo Z Q,Wang L H.2014.Asymbiotic germination and in vitro seedling cultivation of Paphiopedilum emersonii[J].Guizhou Forestry Science and Technology,42(3):34-38.
杨燕萍,徐晓薇,姚丽娟,林霞,张庆良,张旭乐,黄建.2011.亨利兜兰茎尖诱导与离体快繁试验[J].农业科技通讯,(4):53-55.Yang Y P,Xu X W,Yao L J,Lin X,Zhang Q L,Zhang X L,Huang J.2011.Shoot tips culture and in vitro propagation of Paphiopedilum henryanum[J].Bulletin of Agricultural Science and Technology,(4):53-55.
叶维雁,郭晓月,刘惠民,王连春,刘鹏,吴海波.2015.葡萄柚种子无菌苗组培快繁体系的建立[J].广西植物,35(6):891-898.Ye W Y,Guo X Y,Liu H M,Wang L C,Liu P,Wu H B.2015.Establishment of rapid propagation for sterile seedlings of grapefruit by tissue culture[J].Guihaia,35(6):891-898.
尤佳妍,张毓,刘岩,程瑾,潘会堂,张启翔.2014.小叶兜兰的种子发育和无菌萌发[J].植物生理学报,50(3):275-282.You J Y,Zhang Y,Liu Y,Cheng J,Pan H T,Zhang Q X.2014.Seed development and asymbiotic germination of Paphiopedilum barbigerum Tang et Wang[J].Plant Physiology Journal,50(3):275-282.
张娟娟,严宁,胡虹.2013.三种兜兰属植物种子发育过程及其与无菌萌发的关系[J].植物分类与资源学报,35(1):33-40.Zhang J J,Yan N,Hu H.2013.The seed development of three Paphiopedilum species in relation to asymbiotic germination[J].Plant Diversity and Resources,35(1):33-40.
曾宋君,陈之林,段俊.2006.带叶兜兰的无菌播种和离体快速繁殖[J].植物生理学通讯,42(2):247.Zeng S J,Chen Z L,Duan J.2006.Asepsis sowing and in vitro propagation of Paphiopedilum hirsutissimum Pfitz[J].Plant Physiology Communications,42(2):247.
曾宋君,陈之林,吴坤林,段俊.2007.兜兰无菌播种和组织培养研究进展[J].园艺学报,34(3):793-796.Zeng S J,Chen Z L,Wu K L,Duan J.2007.A resume of germination and mericlone of Paphiopediium[J].Acta Horticulturae Sinica,34(3):793-796.
周丽,邓克云,徐正海.2013.“金州报春”兜兰的非共生萌发与快速繁殖[J].植物生理学报,49(11):1250-1254.Zhou L,Deng K Y,Xu Z H.2013.In vitro asymbiotic germination and propagation of Paphiopedilum primulinum'Jinzhoubaochun'[J].Plant Physiology Journal,49(11):1250-1254.
祝剑峰,李芬,袁宇明,王丽珍.2015.鸢尾组织培养快速繁殖技术研究[J].江西农业学报,27(5):25-28.Zhu J F,Li F,Yuan Y M,Wang L Z.2015.Research on tissue culture and rapid propagation of Iris[J].Acta Agriculturae Jiangxi,27(5):25-28.
Pierik R L M,Sprenkels P A,Van Der Harst B,Van Der Meys Q G.1988.Seed germination and further development of plantlets of Paphiopedilum ciliolare Pfitz.in vitro[J].Scientia Horticulturae,34(1):139-153.
Zeng S J,Chen Z L,Wu K L,Zhang J X,Bai C K,Da Silva J A T,Duan J,Zeng S J.2011.Asymbiotic seed germination,induction of calli and protocorm-like bodies,and in vitro seedling development of the rare and endangered Nothodoritis zhejiangensis Chinese orchid[J].Hort Science,46(3):460-465.
Zeng S J,Wu K L,Teixeira da silva J A,Zhang J X,Chen Z L,Xia N H,Duan J.2012.Asymbiotic seed germination,seedling development and reintroduction of Paphiopedilum wardii Sumerh,an endangered terrestrial orchid[J].Scientia Horticulturae,138(5):198-209.
陈宇勒.2009.兰花繁育技术图解[M].广州:广东科技出版社.Chen Y L.2009.Orchid Breeding Technology Illustration[M].Guangzhou:Guangdong Science and Technology Publishing House.
陈之林,叶秀粦,梁承邺,段俊.2004.杏黄兜兰和硬叶兜兰的种子试管培养[J].园艺学报,31(4):540-542.Chen Z L,Ye X L,Liang C Y,Duan J.2004.Seed germination in vitro of Paphiopedilum armeniacum and P.micranthum[J].Acta Horticulturae Sinica,31(4):540-542.
崔秋华,孙永玉,李昆,周志美,段明伦.2012.不同因子对齿瓣石斛增殖系数和平均苗高的影响[J].中南林业科技大学学报,32(4):200-203.Cui Q H,Sun Y Y,Li K,Zhou Z M,Duan M L.2012.Effects of different factors on proliferation coefficient of Dendrobium devonianum and average seedling height[J].Journal of Central South University of Forestry&Technology,32(4):200-203.
丁长春,夏念和.2009.麻栗坡兜兰的无菌播种与快速繁殖[J].植物生理学通讯,45(2):1201-1202.Ding C C,Xia N H.2009.Aseptic sowing and rapid propagation of Paphiopedilum malipoense S.C.Chen&Z.H.Tsi in vitro[J].Plant Physiology Communications,45(2):1201-1202.
丁长春,虞泓,刘方媛.2004.影响杏黄兜兰种子萌发的因素[J].云南植物研究,26(6):673-677.Ding C C,Yu H.Liu F Y.2004.Factors affecting the germination of Paphiopedilum armeniacum[J].Acta Botanica Yunnanica,26(6):673-677.
李慧敏,杨冠海,李明静,闻永慧,张青华,李枝林.2013.白芨瓶内假鳞茎诱导研究[J].南方农业学报,44(10):1607-1612.Li H M,Yang G H,Li M J,Wen Y H,Zhang Q H,Li Z L.2013.Bletilla striata pseudobulb induction in hottle[J].Journal of Southern Agriculture,44(10):1607-1612.
刘仲健,刘可为,陈利君,雷嗣鹏,李利强,施晓春,黄来强.2006.濒危物种杏黄兜兰的保育生态学[J].生态学报,26(9):2791-2800.Liu Z J,Liu K W,Chen L J,Lei S P,Li L Q,Shi X C,Huang L Q.2006.Conservation ecology of endangered species Paphiopedilum armeniacum(Orchidaceae)[J].Acta Ecologica Sinica,26(9):2791-2800.
罗毅波,贾建生,王春玲.2003.初论中国兜兰属植物的保护策略及其潜在资源优势[J].生物多样性,11(6):491-498.Luo Y B,Jia J S,Wang C L.2003.Conservation strategy and potential advantages of the Chinese Paphiopedilum[J].Biodiversity Science,11(6):491-498.
秦丽凤,石贵玉.2013.外源激素对杂交兰组培快繁的效应[J].南方农业学报,44(9):1435-1438.Qin L F,Shi G Y.2013.Effects of exogenous growth regulators on rapid propagation of Cymbidium hybridum×Faberi[J].Journal of Southern Agriculture,44(9):1435-1438.
史军,程瑾,罗敦,上官法治,罗毅波.2007.利用传粉综合征预测:长瓣兜兰模拟繁殖地欺骗雌性食蚜蝇传粉[J].植物分类学报,45(4):551-560.Shi J,Cheng J,Luo D,Shangguan F Z,Luo Y B.2007.Pollination syndromes predict brood-site deceptive pollination by female hoverflies in Paphiopedilum dianthum(Orchidaceae)[J].Acta Phytotaxonomica Sinica,45(4):551-560.
孙晓颖.2014.五种野生兜兰植物菌根真菌多样性研究[D].北京:北京林业大学.Sun X Y.2014.Diversity of mycorrhizal fungi isolated from five species of the genus Paphiopedilum[D].Beijing:Beijing Forestry University.
田凡,姜运力,罗在柒,王莲辉.2014.白花兜兰种子无菌萌发及试管成苗技术研究[J].贵州林业科技,42(3):34-38.Tian F,Jiang Y L,Luo Z Q,Wang L H.2014.Asymbiotic germination and in vitro seedling cultivation of Paphiopedilum emersonii[J].Guizhou Forestry Science and Technology,42(3):34-38.
杨燕萍,徐晓薇,姚丽娟,林霞,张庆良,张旭乐,黄建.2011.亨利兜兰茎尖诱导与离体快繁试验[J].农业科技通讯,(4):53-55.Yang Y P,Xu X W,Yao L J,Lin X,Zhang Q L,Zhang X L,Huang J.2011.Shoot tips culture and in vitro propagation of Paphiopedilum henryanum[J].Bulletin of Agricultural Science and Technology,(4):53-55.
叶维雁,郭晓月,刘惠民,王连春,刘鹏,吴海波.2015.葡萄柚种子无菌苗组培快繁体系的建立[J].广西植物,35(6):891-898.Ye W Y,Guo X Y,Liu H M,Wang L C,Liu P,Wu H B.2015.Establishment of rapid propagation for sterile seedlings of grapefruit by tissue culture[J].Guihaia,35(6):891-898.
尤佳妍,张毓,刘岩,程瑾,潘会堂,张启翔.2014.小叶兜兰的种子发育和无菌萌发[J].植物生理学报,50(3):275-282.You J Y,Zhang Y,Liu Y,Cheng J,Pan H T,Zhang Q X.2014.Seed development and asymbiotic germination of Paphiopedilum barbigerum Tang et Wang[J].Plant Physiology Journal,50(3):275-282.
张娟娟,严宁,胡虹.2013.三种兜兰属植物种子发育过程及其与无菌萌发的关系[J].植物分类与资源学报,35(1):33-40.Zhang J J,Yan N,Hu H.2013.The seed development of three Paphiopedilum species in relation to asymbiotic germination[J].Plant Diversity and Resources,35(1):33-40.
曾宋君,陈之林,段俊.2006.带叶兜兰的无菌播种和离体快速繁殖[J].植物生理学通讯,42(2):247.Zeng S J,Chen Z L,Duan J.2006.Asepsis sowing and in vitro propagation of Paphiopedilum hirsutissimum Pfitz[J].Plant Physiology Communications,42(2):247.
曾宋君,陈之林,吴坤林,段俊.2007.兜兰无菌播种和组织培养研究进展[J].园艺学报,34(3):793-796.Zeng S J,Chen Z L,Wu K L,Duan J.2007.A resume of germination and mericlone of Paphiopediium[J].Acta Horticulturae Sinica,34(3):793-796.
周丽,邓克云,徐正海.2013.“金州报春”兜兰的非共生萌发与快速繁殖[J].植物生理学报,49(11):1250-1254.Zhou L,Deng K Y,Xu Z H.2013.In vitro asymbiotic germination and propagation of Paphiopedilum primulinum'Jinzhoubaochun'[J].Plant Physiology Journal,49(11):1250-1254.
祝剑峰,李芬,袁宇明,王丽珍.2015.鸢尾组织培养快速繁殖技术研究[J].江西农业学报,27(5):25-28.Zhu J F,Li F,Yuan Y M,Wang L Z.2015.Research on tissue culture and rapid propagation of Iris[J].Acta Agriculturae Jiangxi,27(5):25-28.
Pierik R L M,Sprenkels P A,Van Der Harst B,Van Der Meys Q G.1988.Seed germination and further development of plantlets of Paphiopedilum ciliolare Pfitz.in vitro[J].Scientia Horticulturae,34(1):139-153.
Zeng S J,Chen Z L,Wu K L,Zhang J X,Bai C K,Da Silva J A T,Duan J,Zeng S J.2011.Asymbiotic seed germination,induction of calli and protocorm-like bodies,and in vitro seedling development of the rare and endangered Nothodoritis zhejiangensis Chinese orchid[J].Hort Science,46(3):460-465.
Zeng S J,Wu K L,Teixeira da silva J A,Zhang J X,Chen Z L,Xia N H,Duan J.2012.Asymbiotic seed germination,seedling development and reintroduction of Paphiopedilum wardii Sumerh,an endangered terrestrial orchid[J].Scientia Horticulturae,138(5):198-209.