西藏虎头兰高效植株再生体系的研究
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摘要
为建立西藏虎头兰(Cymbidium tracyanum)的高效快速繁殖体系,该研究以野生西藏虎头兰种子为外植体,通过分析不同基本培养基和植物激素配比对原球茎诱导、增殖和分化的影响,以及光照时间和培养温度对试管苗生长的影响,筛选出适宜西藏虎头兰植株高效再生的条件。结果表明:适宜西藏虎头兰生长的基本培养基为1/2 MS;种子萌发和原球茎诱导的最适培养基为1/2 MS+1.0 mg·L~(-1)6-BA+0. 5 mg·L~(-1)NAA,培养50 d后,有95.00%的种子发育成原球茎;原球茎增殖的最适培养基为1/2 MS+2.0 mg·L~(-1)NAA,培养30 d,增殖倍数为4.25;原球茎的最适分化培养基为1/2 MS+2.0 mg·L~(-1)NAA+60 g·L~(-1)土豆泥+0.5g·L~(-1)活性炭,培养10 d,不定芽发生率为98.33%,培养40 d,幼苗生根率为94.67%;试管苗在温度20℃、光照时间12 h·d~(-1)、光照强度2 000 lx的条件下培养,苗长势好,叶片生理性焦尖发生率仅为3.33%;以腐殖土作为栽培基质,试管苗的移栽成活率为97.78%。该研究结果为保护西藏虎头兰野生资源和工厂化育苗提供了科学依据和技术支持。
In order to establish a highly efficient plant regeneration system,Cymbidium tracyanum was selected as explant to screen out the conditions to regenerate with highly efficiency,by evaluating the effects of basic medium and hormone combination on the plant regeneration of C. tracyanum,and the effects of illumination time and culture temperature on the growth of plantlets were also studied. The results were as follows: The optimum basic medium for C. tracyanum growth was 1/2 MS. The optimal medium for protocorm induction was 1/2 MS+1.0 mg·L~(-1)6-BA+ 0.5 mg·L~(-1) NAA,95.00% of the seeds developed into the protocorms after cultured for 50 d. The optimal medium for protocorm proliferation was 1/2 MS+2.0 mg·L~(-1) NAA,the maximum proliferation multiple of protocorm reached 4.25 after cultured for 30 d. The optimal medium for protocorm differentiation was 1/2 MS+2.0 mg·L~(-1) NAA+60 g·L~(-1) mashed potato+0.5 g·L~(-1) acticarbon,the induction rate of indefinite bud was 98.33% after cultured for 10 d,the rooting rate was 94.67% after cultured for 40 d. In the process of plantlets subculture,temperature of 20 ℃,illumination time of 12 h·d~(-1) and illumination intensity of 2 000 lx were suited for the growth of plantlets,the physiological scorch rate of leaf tip was3.33%. Using humus as cultivation substrate,the transplanting survival rate of plantlets was 97.78%. This study provides an effective way for keeping good varieties of features and rapid propagation of C. tracyanum,at the same time,helps to solve some theoretical problems in factory production of C. tracyanum.
In order to establish a highly efficient plant regeneration system,Cymbidium tracyanum was selected as explant to screen out the conditions to regenerate with highly efficiency,by evaluating the effects of basic medium and hormone combination on the plant regeneration of C. tracyanum,and the effects of illumination time and culture temperature on the growth of plantlets were also studied. The results were as follows: The optimum basic medium for C. tracyanum growth was 1/2 MS. The optimal medium for protocorm induction was 1/2 MS+1.0 mg·L~(-1)6-BA+ 0.5 mg·L~(-1) NAA,95.00% of the seeds developed into the protocorms after cultured for 50 d. The optimal medium for protocorm proliferation was 1/2 MS+2.0 mg·L~(-1) NAA,the maximum proliferation multiple of protocorm reached 4.25 after cultured for 30 d. The optimal medium for protocorm differentiation was 1/2 MS+2.0 mg·L~(-1) NAA+60 g·L~(-1) mashed potato+0.5 g·L~(-1) acticarbon,the induction rate of indefinite bud was 98.33% after cultured for 10 d,the rooting rate was 94.67% after cultured for 40 d. In the process of plantlets subculture,temperature of 20 ℃,illumination time of 12 h·d~(-1) and illumination intensity of 2 000 lx were suited for the growth of plantlets,the physiological scorch rate of leaf tip was3.33%. Using humus as cultivation substrate,the transplanting survival rate of plantlets was 97.78%. This study provides an effective way for keeping good varieties of features and rapid propagation of C. tracyanum,at the same time,helps to solve some theoretical problems in factory production of C. tracyanum.
引文
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LAN YT,WU TG,LIU SY,et al.,2010.Study on tissue culture of wild Cymbidium tracyanum L.Castler[J].J Fujian For Sci Technol,37(1):77-79.[蓝玉甜,吴天贵,刘世勇,等,2010.野生西藏虎头兰组培技术研究[J].福建林业科技,37(1):77-79.]
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LIU SS,CHEN J,GUO SX,2015.Review on germination of Orchid seeds[J].Seed,34(6):43-49.[刘思思,陈娟,郭顺星,2015.兰科植物种子萌发的研究进展[J].种子,34(6):43-49.]
LI JW,ZHANG SB,2016.Differences in the responses of photosystems I and II in Cymbidium sinense and C.tracyanum to long-term chilling stress[J].Front Plant Sci,doi:10.3389/fpls.2015.01097.
LI JW,CHEN XD,HU XY,et al.,2018.Comparative physiological and proteomic analyses reveal different adaptive strategies by Cymbidium sinense and C.tracyanum to drought[J].Planta,247(1):69-97.
QIU Y,GONG N,ZHANG KY,2010.Seed germination and the protocorm multiplication of Anoectochilus roxburghii[J].Guihaia,30(4):555-559.[邱玥,龚宁,张奎一,2010.金线兰种子萌发及其原球茎的增殖培养[J].广西植物,30(4):555-559.]
WANG LH,JIANG YL,YU JY,et al.,2009.Tissue culture and rapid propagation of Cymbidium tracyanum L.Castler.[J].Plant Physiol Comm,45(1):51-52.[王莲辉,姜运力,余金勇,等,2009.西藏虎头兰的组织培养与快速繁殖[J].植物生理学通讯,45(1):51-52.]
WANG XL,2018.Evaluation of endangered grade of wild Orchidaceae in Tibet[J].Tibet Sci Technol,(1):65-67.[王喜龙,2018.西藏野生兰科植物濒危等级评价[J].西藏科技,(1):65-67.]
WANG ZH,2003.Diagnosis and treatment of physiological scorch of leaf tip of orchid[J].Flower Plant Penjing,(6):32.[王志红,2003.兰叶生理性焦尖的诊治[J].花木盆景,(6):32.]
WU YG,2003.Diagnosis and treatment of bacterial scorch of leaf tip of orchid[J].Flower Plant Penjing,(6):32.[吴亚光,2003.兰叶细菌性焦尖的诊治[J].花木盆景,(6):32.]
YAO SC,LING ZZ,LAN ZZ,et al.,2012.Study on seed germination and the protocorm multiplication of Phaius tankervillese[J].Acta Bot Boreal-Occident Sin,32(7):1474-1479.[姚绍嫦,凌征柱,蓝祖栽,等,2012.鹤顶兰种子萌发及原球茎增殖培养研究[J].西北植物学报,32(7):1474-1479.]
CHEN HM,LüFB,LI Z,et al.,2015.The tissue culture and rapid propagation of Cymbidium tracyanum L.Castler.[D]//Chinese Society for Horticultural Science.Advances in Ornamental Horticulture of China.Beijing:China Forestry Publishing House:253-255.[陈和明,吕复兵,李佐,等,2015.西藏虎头兰组培快繁技术研究[D]//中国园艺学会.中国观赏园艺研究进展.北京:中国林业出版社:253-255.]
Institute of Botany,the Chinese Academy of Sciences,2013.List of rare and endangered plants in China[DB/OL].http://rep.iplant.cn/protlist.[中国科学院植物研究所,2013.中国珍稀濒危植物名录[DB/OL].http://rep.iplant.cn/protlist.]
KUANG ML,ZHANG BS,2015.Physiological response to high light in Cymbidium tracyanum and C.sinense[J].Plant Divers Resour,37(1):55-62.[匡美龄,张石宝,2015.西藏虎头兰和墨兰对强光的生理响应[J].植物分类与资源学报,37(1):55-62.]
LAN YT,WU TG,LIU SY,et al.,2010.Study on tissue culture of wild Cymbidium tracyanum L.Castler[J].J Fujian For Sci Technol,37(1):77-79.[蓝玉甜,吴天贵,刘世勇,等,2010.野生西藏虎头兰组培技术研究[J].福建林业科技,37(1):77-79.]
LI HM,2014.Advances in studies of tissue culture and rapid multiplication of Orchids[J].Agric Res Appl,(1):53-56.[李慧敏,2014.兰花组培快繁研究进展[J].农业研究与应用,(1):53-56.]
LI ZL,YU CX,WANG YY,et al.,2005.Research on aseptic budding and fast propagation of the Cymbidium tracyanum seed[J].Chin Agric Bull,21(8):269-270,281.[李枝林,余朝秀,王玉英,等,2005.野生沉香虎头兰种子无菌萌发及快速繁殖技术研究[J].中国农学通报,21(8):269-270,281.]
LIU SS,CHEN J,GUO SX,2015.Review on germination of Orchid seeds[J].Seed,34(6):43-49.[刘思思,陈娟,郭顺星,2015.兰科植物种子萌发的研究进展[J].种子,34(6):43-49.]
LI JW,ZHANG SB,2016.Differences in the responses of photosystems I and II in Cymbidium sinense and C.tracyanum to long-term chilling stress[J].Front Plant Sci,doi:10.3389/fpls.2015.01097.
LI JW,CHEN XD,HU XY,et al.,2018.Comparative physiological and proteomic analyses reveal different adaptive strategies by Cymbidium sinense and C.tracyanum to drought[J].Planta,247(1):69-97.
QIU Y,GONG N,ZHANG KY,2010.Seed germination and the protocorm multiplication of Anoectochilus roxburghii[J].Guihaia,30(4):555-559.[邱玥,龚宁,张奎一,2010.金线兰种子萌发及其原球茎的增殖培养[J].广西植物,30(4):555-559.]
WANG LH,JIANG YL,YU JY,et al.,2009.Tissue culture and rapid propagation of Cymbidium tracyanum L.Castler.[J].Plant Physiol Comm,45(1):51-52.[王莲辉,姜运力,余金勇,等,2009.西藏虎头兰的组织培养与快速繁殖[J].植物生理学通讯,45(1):51-52.]
WANG XL,2018.Evaluation of endangered grade of wild Orchidaceae in Tibet[J].Tibet Sci Technol,(1):65-67.[王喜龙,2018.西藏野生兰科植物濒危等级评价[J].西藏科技,(1):65-67.]
WANG ZH,2003.Diagnosis and treatment of physiological scorch of leaf tip of orchid[J].Flower Plant Penjing,(6):32.[王志红,2003.兰叶生理性焦尖的诊治[J].花木盆景,(6):32.]
WU YG,2003.Diagnosis and treatment of bacterial scorch of leaf tip of orchid[J].Flower Plant Penjing,(6):32.[吴亚光,2003.兰叶细菌性焦尖的诊治[J].花木盆景,(6):32.]
YAO SC,LING ZZ,LAN ZZ,et al.,2012.Study on seed germination and the protocorm multiplication of Phaius tankervillese[J].Acta Bot Boreal-Occident Sin,32(7):1474-1479.[姚绍嫦,凌征柱,蓝祖栽,等,2012.鹤顶兰种子萌发及原球茎增殖培养研究[J].西北植物学报,32(7):1474-1479.]