非洲菊品种‘靓粉’的离体培养及脱毒技术研究
摘要
非洲菊品种‘靓粉’,花色艳丽、花枝挺拔、切花率高、保鲜期长,具有非常高的观赏价值。非洲菊采用离体培养的方法快速繁殖,不仅能在短时间内大量繁殖优良种苗,而且还避免了分株繁殖和种子繁殖所带来的品种退化问题；在非洲菊生长过程中易受到病毒危害,严重影响到非洲菊切花品质和产量。所以本试验研究的目的是针对‘靓粉’的离体培养与脱毒技术两方面的问题,对保持‘靓粉’的优良种质资源与优良性状的稳定、提高其观赏价值有重要意义。
1以无病虫害的‘靓粉’母株为材料,选取花托、茎尖、叶柄为外植体,以MS为基本培养基,附加不同浓度的6-BA. NAA,诱导外植体产生愈伤组织,继而分化出芽；然后以生根培养基进行诱导生根；后将组培苗移栽于不同基质中,筛选最适合的培养基质。
2对日光温室的生产植株和以花托为外植体的组培苗分别进行番茄黑环病毒(Tomato black ring virus,TBRV)、烟草脆裂病毒(Tobacco rattle virus, TRV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、番茄斑萎病毒(Tomato spotted wilt virus, TSWV) 5种非洲菊易感染病毒进行RT-PCR检测,结果显示两种来源的材料都只携带烟草花叶病毒。
3以‘靓粉’组培苗为材料,采用茎尖组织脱毒、茎尖病毒唑(三氮唑核苷)处理脱毒、花托组织病毒唑处理脱毒、热处理脱毒、病毒唑+热处理脱毒5种脱毒处理。采用RT-PCR技术对处理后组培苗进行检测,试验结果显示,用茎尖组织脱毒和茎尖病毒唑(三氮唑核苷)处理脱毒对非洲菊‘靓粉’脱毒达到100%,花托组织病毒唑5mg/L培养基处理后组培苗脱毒率达到85%,而热处理后组培苗表现出不适应性。
Gerbera Cultivar'Liangfen'is very suitable for viewing and admiring as it has big beautiful color and comely straight stem. And it has high cutting-flower efficiency and long growing time in water. Using the rapid reproduction method of Gerbera culturing not only can reproduce a lot of seed-seedling also can avoid the variety-degeneration problem caused by single-reproduction and seed-reproduction; it could be damaged by viruses easily which seriously affects the quality and yield of cut flow-ers.So the study includes isolated culture and virus-free of Gerbera jiamesonii Bolus. The seeond is Preserving excellent idioplasm resource and stability of fine properties, raising its worth of viewing.
At first, the receptacle, stem apex and petiole of the non-infected Gerbera plants grown in greenhouse were selected in the study. The explants were growing on MS basic medium with 6-BAandNAA in different concentration.6-BA and NAA were us-ed to induce the explant to produce calli and differentiate buds. The leaf of test-tube plantlet is selected as the explant to reduce infection rates. When test-tube plantlet ro-oting, they were planted on different substrates to select the most appropriate.
Secondly, for Gerbera plants grown in greenhouse and test-tube plantlet form receptacle,5 kinds of viruses were detected. They were TBRV, TRV, TMV, CMV and TSWV. Gerbera Cultivar'Liangfen'infect the 5 kinds of viruses easily. The result illus-trated that only could be detected by TMV.
Finally, used heat treatment and medium joining to ribavirin, stem apex and med-ium joining to ribavirin culture, receptacle and medium joining to ribavirin, heat trea-tment culture, and stem apex culture for virus elimination by isolated culture. Among the 5 kin-ds of virus-free, medium joining to ribavirin was used on Gerbera Cultivar 'Liangfen' for the first time.The date illustrated that stem apex culture had a good effect for virus-free of Gerbera Cultivar'Liangfen'.
1以无病虫害的‘靓粉’母株为材料,选取花托、茎尖、叶柄为外植体,以MS为基本培养基,附加不同浓度的6-BA. NAA,诱导外植体产生愈伤组织,继而分化出芽；然后以生根培养基进行诱导生根；后将组培苗移栽于不同基质中,筛选最适合的培养基质。
2对日光温室的生产植株和以花托为外植体的组培苗分别进行番茄黑环病毒(Tomato black ring virus,TBRV)、烟草脆裂病毒(Tobacco rattle virus, TRV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、番茄斑萎病毒(Tomato spotted wilt virus, TSWV) 5种非洲菊易感染病毒进行RT-PCR检测,结果显示两种来源的材料都只携带烟草花叶病毒。
3以‘靓粉’组培苗为材料,采用茎尖组织脱毒、茎尖病毒唑(三氮唑核苷)处理脱毒、花托组织病毒唑处理脱毒、热处理脱毒、病毒唑+热处理脱毒5种脱毒处理。采用RT-PCR技术对处理后组培苗进行检测,试验结果显示,用茎尖组织脱毒和茎尖病毒唑(三氮唑核苷)处理脱毒对非洲菊‘靓粉’脱毒达到100%,花托组织病毒唑5mg/L培养基处理后组培苗脱毒率达到85%,而热处理后组培苗表现出不适应性。
Gerbera Cultivar'Liangfen'is very suitable for viewing and admiring as it has big beautiful color and comely straight stem. And it has high cutting-flower efficiency and long growing time in water. Using the rapid reproduction method of Gerbera culturing not only can reproduce a lot of seed-seedling also can avoid the variety-degeneration problem caused by single-reproduction and seed-reproduction; it could be damaged by viruses easily which seriously affects the quality and yield of cut flow-ers.So the study includes isolated culture and virus-free of Gerbera jiamesonii Bolus. The seeond is Preserving excellent idioplasm resource and stability of fine properties, raising its worth of viewing.
At first, the receptacle, stem apex and petiole of the non-infected Gerbera plants grown in greenhouse were selected in the study. The explants were growing on MS basic medium with 6-BAandNAA in different concentration.6-BA and NAA were us-ed to induce the explant to produce calli and differentiate buds. The leaf of test-tube plantlet is selected as the explant to reduce infection rates. When test-tube plantlet ro-oting, they were planted on different substrates to select the most appropriate.
Secondly, for Gerbera plants grown in greenhouse and test-tube plantlet form receptacle,5 kinds of viruses were detected. They were TBRV, TRV, TMV, CMV and TSWV. Gerbera Cultivar'Liangfen'infect the 5 kinds of viruses easily. The result illus-trated that only could be detected by TMV.
Finally, used heat treatment and medium joining to ribavirin, stem apex and med-ium joining to ribavirin culture, receptacle and medium joining to ribavirin, heat trea-tment culture, and stem apex culture for virus elimination by isolated culture. Among the 5 kin-ds of virus-free, medium joining to ribavirin was used on Gerbera Cultivar 'Liangfen' for the first time.The date illustrated that stem apex culture had a good effect for virus-free of Gerbera Cultivar'Liangfen'.
引文
[1]谭文澄,戴策刚.观赏植物组织培养技术[M].北京：中国林业出版社,1997.
[2]邱强,李贵宝,员连国,等.花卉病虫原色图谱[M].北京：中国建材工业出版社,1998,10：57-58.
[3]李绅崇,李金泽,吴丽芳,蒋亚莲.非洲菊抗疫病切花新品种‘靓粉’.园艺学报,2008,35(3)：466.
[4]Pierik.R.LM.Gerbera plantlets from in vitro cultivated capitum explant[J].Scientia Hort. 1973,(1):117-120.
[5]Murashige.T.Clonal multiplication of Gerbera through tissue culture[J].Hort.Sci.1974,(9): 175-180.
[6]黄济明.儿种花卉的组织培养[J].植物生理学通讯,1983,(3)：45-46.
[7]黄济明,倪跃之,林满红.非洲菊的快速繁殖[J].园艺学报,1987,14(2)：12-128.
[8]黄玉玲,杨宝明.非洲菊的花托离体培养及快繁技术[J].云南农业科技,1998,(5)：33.
[9]王海琴,冯先桔,李丽,罗君琴.非洲菊的组织培养[J].广西农业科学,2005,36(1)：33-34.
[10]王春彦,高年青,张效平,等.非洲菊幼花托离体培养研究[J].江苏农业科学,200(1)：47-49.
[11]杨波,康明.非洲菊组培快繁技术研究[J].湖北农业科学,2000,(2)：48-49.
[12]郑秀芳,李名扬.非洲菊花托培养和植株再生[J].西南农业大学学报,2001,23(2)：171-173.
[13]雷加容,张跃非,刘兴华,邓晓蓉,余敖.非洲菊的组培快繁技术研究[J].绵阳经济技术高等专科学校学报,2002,19(2)：16-17.
[14]王红梅.非洲菊的组培快繁技术研究[J].甘肃农业科技,2000,(10)：42-43.
[15]席梦利,施季森.非洲菊的离体培养及其快速繁殖[J].南京林业大学学报(自然科学版),2003,27(2)：33-36.
[16]张文珠,林德钦,李梅.非洲菊的快繁技术研究[J].福建农业科技,2002,(1)：17-18.
[17]陈海霞,李茂娟.非洲菊不同发育时期的花托诱导愈伤组织的研究[J].广西农业科学,2008,39(1)：1-5.
[18]杨桂芬,王毓,丁红光.非洲菊组织培养与快速繁殖[J].植物生理学通讯,2000,36(4)：333.
[19]郑秀芳,王桔红,李名扬.影响非洲菊离体培养器官分化的因素[J].江苏林业科技,2002,29(1)：29-31.
[20]鲁雪华,林勇,郭文杰.非洲菊小花托的离体培养[J].亚热带植物通讯,1996,25(2)：21-24.
[21]杨光穗.非洲菊组织培养研究进展[J].热带农业科学,2003,23(1)：57.
[22]刘丽荣,苏荣德,李忠利.非洲菊组织培养繁殖的试验研究[J].辽宁农业职业技术学院学报,2002,4(3)：13-15.
[23]陈发棣,陈滨.非洲菊组织培养中La3+的应用初探[J].园艺学报,2002,29(4)：383-385.
[24]倪丹,杨世湖,徐世清等.非洲菊组培快繁的优化[J].细胞生物学杂志,2002,24 (5)：316-319.
[25]李启任,王云经,夏从龙.非洲菊组培快繁技术研究[J].云南大学学报(自然科学版)1998,20(生物学专辑),560-563
[26]石文山,李树丽,胡敬宜,滕娜.非洲菊的离体培养和快速繁殖技术研究[J].热带农业科学,2006,(12)：44-46.
[27]许彩霞,孙成林,薛炜等.非洲菊组织培养离体快速繁殖[J].内蒙古林业科技,2001,(增刊).
[28]王玉芹,王喜军.非洲菊的组织培养[J]。中国林副特产,2000,(2)：25.
[29]刘福平,林丽仙,邹小鲁等.非洲菊组织培养(简报).亚热带植物通讯,2000,29(2)：52.
[30]陈发棣,李倩中,房伟民等.两个非洲菊品种叶片的离体培养[J].江苏林业科技,1998,25(增刊)：174-176.
[31]曾长立,陶婷,陈禅友.非洲菊的组织培养与快速繁殖[J].江汉大学学报(自然科学版),2005,33(4)：36-38.
[32]戴云新,张健,李敏,李玉娟.不同外植体和激素对非洲菊愈伤组织诱导及芽分化的影响[J].浙江农业科学,2009(4)：695-696.
[33]曹征宇,严志衡,贾明姚愚,顾韵莉,虞冠军.非洲菊离体快速繁殖技术[J].上海交通大学学报(农业科学版),2007,25(4)：395-398.
[34]杨继涛,邹志荣,张素勤,等.植物激素对非洲菊花托愈伤组织诱导的研究[J].西北农业学报,2003,12(3)：133-135.
[35]鲁雪华,郭文杰,林勇.几种因素对非洲菊离体培养再生植株的影响[J].植物生理学通讯,1999,35(5)：372-374.
[36]刘晓燕.非洲菊离体组织培养与快速繁殖[J].耕作与栽培,2001(3)：21.
[37]杨华,阮颖,刘春林等.非洲菊离体再生体系的建立[J].湖南农业科学,2002,(2)：53-54.
[38]徐立,李克烈,李志英等.非洲菊的组织培养和快速繁殖[J]热带农业科学,2000,20(2)：26-27.
[39]张文株,林德钦,李梅.非洲菊的快繁技术[J].福建农业科技,2002(1)：17-18.
[40]王春,余有祥.盆栽非洲菊的组织培养及快速繁殖[J].浙江林业科技,2001,21(3)：30-35
[41]张素勤,邹志荣,耿广东,张亚娟.非洲菊组织培养抑制褐变现象的研究[J].贵州农业科学,2007,35(2)：56.
[42]张仲凯,李毅.云南植物病毒.北京：科学出版社,2001.
[43]除东平,谢联辉.黄瓜花叶病毒亚组研究进展[J].福建农业大学学报,1998,27(1)：82-91.
[44]王健华,王运勤,吉训聪,刘志昕,郑服丛.植物病毒检测技术研究进展[J].热带农业科学,2005,25(3)：71-74.
[45]Derrick K S. Quantitative assay for plant viruses using serologically specific electron microscopy. Virology,1973, (56):652-657.
[46]Louro, D, L esemann D E. Use of protein A-gold complex for specific labeling of antibodies bound to plant viruses-Viral antigens in suspensions. Journal of Virological Methods,1984, (9):107-122.
[47]陈继峰,赵军营,李绍华.果树病毒检测方法及其应用[J].河北林果研究,2005,20(2)：167-173.
[48]陈枝楠,卢高泽,卢俭.PCR应用技术进展简介[J].植物病理学报,1997,27(3)：198-200.
[49]D Colinet, J Kummert, P Lepoivre, et al. Identification of distinct potyviruses in mixedly-infected sweetpotato by the polymerase chain reaction with degenerate primers [J].Phytopathology,1993,84 (1):65-69.
[50]张晶红,徐启江,陈典.唐菖蒲病毒病原鉴定的研究进展[J].黑龙江农学,2008,(6)：159-162.
[51]兰玉菲,郗丽君,张德满等.植物病毒检测技术[J].山东农业科学,2006,5：58-62.
[52]王升吉,尚佑芬,赵玖华等.葡萄卷叶病毒主要检测技术比较[J].山东农业科学,2008,3：95-98.
[53]刘文洪,洪健,陈集双等.百合黄瓜花叶病毒及其检测[J].植物保护,2006,32(1).73-76.
[54]梁巧兰,魏列新,徐秉良.侵染观赏百合的黄瓜花叶病毒血清学检测方法研究[J].甘肃农业大学学报.2009,44(3)：97-101.
[55]Devergne J C,Vardin L,Burckard,J,et al. Compari-song of derect and indirect EL ISA for detecting anti-gencally related cucumovires [J]. Virol method,1981,3 (4):193-199.
[56]杨超,孙秀东.应用RT—PCR检测辣椒上的黄瓜花叶病毒[J].山东农业科学,2009,2：94-96
[57]S. K. Raj,A. Srivastava,G. Chandra,et al. Characterization of cucumber mosaic virus isolate infecting Gladiolus cultivars and comparative evaluation of serological and molecular methods for sensitive diagnosis[J]. Curr. Sci.2002,83:1132-1137.
[58]FANQUETCM, MAYOM A, MAN ILOFF J, et al. Virus Taxonomy-8 th reports of the International Committee on Taxonomy of Viruses [R]. Netherland:Elsevier, Academic Press, 2005.
[59]Brittlebank C C. Tomato diseases. Journal of Agriculture, Victoria 1919,27:231-235.
[60]洪霓.番茄斑萎病毒的检疫技术[J].植物检疫,2006,20(6)：389-392.
[61]Boonham N, P Smith, KWalsh, et al. The detection of Tomato spotted wilt virus (TSWV) in individual thrip s using real time fluorescentRT-PCR (TaqMan). J VirolMethods,2002,101: 37-48.
[62]韩贻仁.分子细胞生物学(第二版)[M].北京：科学出版社,2001.
[63]Ie T S. CMI/AAB Descr PI. Viruses,1970,(39),4pp.
[64]于翠,杨翠云,印丽萍.番茄斑萎病毒--一种值得重视的植物检疫病毒[J].植物检疫,2006增刊,20：47-50.
[65]张仲凯,方琦,丁铭等.侵染烟草的番茄斑萎病毒(TSWV)电镜诊断鉴定[J].电子显微学报,2000,19(3)：339-340.
[661KURSTAK.植物病毒比较诊断指南[M].裴美云译.北京：农业出版社,1993：335-339.
[67]LAWSON R H, D I ENELTM M, HSU H T. The cytopathology of Tospovirus2Host2Plant Interactions [J].Acta Hoticulturae,1996,431:267-290.
[68]Ie T S. CMI/AAB Descr PI. Viruses,1970,(39),4pp.
[69]GonsalvesD, Trujillo E E. Tomato spotted wilt virus in papaya and detection of the virus by EL ISA. Plant Dis.1986,70:501-506.
[70]Wang M, Gonsalves D. EL ISA detection of various tomato spotted wilt virus isolates using specific antisera to structural proteins of the virus. Plant Dis.1990,74,154-158.
[71]Sherwood J L, Sanbom M R, Keyser G C, et al. Use of monoclonal antibodies in detection of tomato spotted wilt virus. Phytopathol,1989,79:61-64.
[72]Hugenot C, Van Den Dobbelsteen, G de Haan P. Detection of tomato spotted wilt virus using mono-clonal antibodies and ribop robes Arch Virol,1990,110,47-62.
[73]郑元仙,刘雅婷.番茄斑萎病毒属病毒检测技术研究进展[J].云南农业大学学报,2009年,24(4),607-613.
[74]BYOUNG-CHEORL K IY, MOLLYM J. Genetics of Plant Virus Resistance [J]. Annual Review of Phytopathology,2005,43:581-621.
[75]RESENDE EW, KITAJ IMA RW, PETERS G D. SerologicalDifferentiation of 20 Isolates of Tomato Spotted wilt Virus [J]. Journal of General Virology,1990,71:2801-2807.
[76]GONSALVESW M D. EL ISA Detection of Various Tomato Spotted wilt Virus Isolates using Specific Antisera to Structural Proteins of the Virus [J]. Plant Disease,1990,74:154-158.
[77]Rice D J, German T L, Mau R F L, et al. Dot blot detection of Tomato spotted wilt virus RNA in p lant and thrip s tissues by cDNA clones. Plant Disease,1990,74:274-276.
[78]Ronco A E,Dal Bo E, Ghiringhelli P D, et al. Cloned cDNA p robes for the detection tomato spotted wilt virus. Phytopathol,1989,79:69-74.
[79]WEEKES R J, MUMFORD R A, BARKER I. Diagnosis of Tospovirus by Reverse-transcription Polymerase Chain Reaction [J]. Acta Hoticultures,1996,431:159-166.
[80]MUMFORD R A, BARKER I, WOOD K R. An Improved Method for the Detection of Tospoviruses Using the polymerase Chain Reaction [J]. Journal of VirologicalMethods, 1996,57:109-115.
[81]CORTEZI J S, WONGJKAEW K S, PEREIRA A M.Identification and Characterization of a Novel Tospovirus Species using a New RT-PCR app roach [J]. Archivesof virology,2001, 146:265-278.
[83]H IGUCH IR, FOCKLER C, DOLL INGER G. Kinetic PCR Analysis:Real2timeMonitoring of DNA Amp lification Reactions [J]. Biotechnology,1993,11:1026.
[82]MORR IS J. Diagnostic Protocols for Regulated Pests Protocoles de Diagnostic Pourles Organismes Reglementes [J]. OEPP/EPPO Bulletin,2004,34 (2):271-279.
[84]CASSI E A, ROBERTSA R G D, L ISA A. Real—timeRT2PCR Fluorescent Detection of Tomato Spotted wilt Virus [J]. Journal of Virological Methods,2000,88:1-8.
[85]付鸣佳,高乔婉,范怀忠.烟草花叶病毒株系鉴定研究进展[J].华南农业大学学报,1997,18(4)：113-117.
[86]张振臣,张丽芳,乔奇,等.河南省地黄病毒病初步鉴定[J].植物病理学报,2004,34(5)：395--399.
[87]王敏,李明福,黄璐琦,等.栽培地黄(Rehmannia glutinosa Liboseh)普遍感染TMV和CMV[J].植物病理学报,2006,36(2)：189-192.
[88]Harrison BD and Nixon HL. Separalon and properties of particles of tobacco rattle virus with different lengths. [J].Gen Microhiol.1958,21:569-581.
[89]Harrison BD and Woods RD. Serotypes and particle dimensions of tobacco rattle viruses from Europe and America.Virology.1966,28:610-620.
[90]马新颖,汪琳,任鲁风,等.应用基因芯片检测烟草脆裂病毒.植物病理学报,2007,37(4)：352-355.
[91]Brunt AA,Crabtree K,Dallwitz MJ,et al.Viruses ofplants. CAB International [M]. Walling ford,UK:1996.
[92]Wellink J,Le Gall O,Sanfacon H,et al.Family Comoviridae.In:Fauquet CM(ed)The Seventh Report of the International Committee on Taxonomy of Viruses[M],Academic Press,San Diego,CA,pp 2000.
[93]Murant A F. Seed and pollen transmission of nematode-borne viruses.Seed Science and Technology,1983,11:973-987.
[94]杜更新,曹新民,李尉民.番茄黑环病毒(TBRV)初步鉴定报告.中国烟草,1992(2)：28.
[95]蔡汉权,李粉玲,林珊珊.花卉脱毒快繁技术研究进展,江西科学,2006,4(24),124-127.
[96]葛胜娟.植物组织培养中的快繁与脱毒技术及其应用[J].中国农学通报,2005,5(21),104-108.
[97]赵军良.植物茎尖培养与无毒种苗生产[J].北方园艺,1995(6)：10-11.
[98]曹为玉,郑燕棠,张福庆等.葡萄茎尖脱毒培养和快速繁殖[J].华北农学报,1993,88(2)：69-72
[99]席梦利,王节萍,章静娟等.宜兴百合脱毒技术[J].江苏农业学报,2001,17(1)：49-51.
[100]张玉清,罗晓芳,田砚亭.葡萄微茎尖培养和葡萄扇叶病毒的ELISA和探针检测[J].北京林业大学学报,1998,20(4)：54-58.
[101]朱德蔚.组织培养与脱毒快繁技术[M].北京：国科学技术出版社,2001。
[102]王永伟,王慧霞,贺丹,何松林.观赏植物病毒病害及病毒脱除研究现状与发展趋势中国农学通报.2008,24(5)：313-317.
[103]谢嘉华,袁建军,花卉脱毒培养与优质种苗生产[J].生物学杂志,2001,18(5)：37-38.
[104]邓衍明,褚芳玲,华如枝.滁菊组织培养脱病毒技术研究[J].安徽农业科学,2007,35(14)：4130,4158.
[105]赵祝成,陈燕贤中国水仙脱毒种苗培育研究取得重大突[J].中国花卉园艺,2003(1)：331.
[106]符国芳,李青.植物组织培养脱毒方法综述.2007,34(3)：255-258.
[107]李文安.业生物工程[M].北京：化学工业出版社,1998：4.
[108]Lizarrage C,Fermandez-Northcote E N1 Detection of potato viruses X and Yin sap extracts by a modified indirect enzyme-linked immunosorbent assay on nitrocellulose membranes (NCM-EL ISA) [J]. Plant Disease,1989,73:11-141
[109]谢嘉华,袁建军.中国水仙(Narcissustazetta varlchinese)的组织培养[J].生物学杂志,2002,19(3)：30.
[110]Engelmann F.Plant cryopreservation:progress and prospects [J]. In Vitro Cellular& Developmental Biology,2004,40:427-433.
[111]蔡汉权,李粉玲,林珊珊.花卉脱毒快繁技术研究进展[J].江西科技,2006(2)：124-127
[112]Helliot B,Panis B,Poumay Y,et al.Cryopreservation for the elimination of cucumber mosaic and banana streak viruses from banana (Musa spp.)[J]. Plant Cell Rep,2002, 20:1117-1122.
[113]高遐红,李梅.提高草莓茎尖组织培养脱毒效果研究[J].中国果树,1994,(2)：5-6.
[114]周厚成,何水涛.草莓病毒病研究进展[J].果树学报,2003,(5)：421-426.
[115]高庆玉,李光裕,周恩.关于草莓脱毒技术的研究[J].东北农学院学报,1993,(3)：231-23.
[116]Thomson A J, et al.The evaluation of potato somaclones. In:Semal Jed. Somaclonal Variations and Crop Improvement. Martnus Nijhoff Publishers. Dordrecht.1986:236-243.
[117]孔宝华,蔡红,陈海如等.花卉病毒病及防治[M].北京：中国农业出版社,2002：119-121.
[118]Sanchez Navarro J A. Comparative analysis of ELISA, nonradioactive molecular hybridization and PCR for the detection of Prunus necrotic ring spot virus in herbaceous and prunus host[J]. Plant Pathol,1998,47:7802786.
[119]Sanchez Navarro J A. Simultaneous detection and identificationof eight stone fruit viruses by one-step RT-PCR,European[J] Journal of Plant Pathology,2005,111:77284.
[120]兰平,李文风等.热处理结合茎尖培养去除甘薯丛枝病植原体[J].西北农林科技大学学 报,2001,6：1-4.
[121]F.A.Hakkaart,G.Hartel.Virus eradication from some Pelargonium zonale cultivars by meristem-tipculture[J]. European Journal of Plant Pathology,1979,85:3-46
[122]R.Ram,N.Verma.Indexing and production of virus-free chrysanthemums.Biologia plantarum,2005,49(1):149-152
[123]G.A.Manganaris,A.S.Economou,et al.Elimination of PPV and PNRSV through thermotherapyand meristem-tip culture in nectarine.Plant Cell Rep,2003,22:195-200.
[2]邱强,李贵宝,员连国,等.花卉病虫原色图谱[M].北京：中国建材工业出版社,1998,10：57-58.
[3]李绅崇,李金泽,吴丽芳,蒋亚莲.非洲菊抗疫病切花新品种‘靓粉’.园艺学报,2008,35(3)：466.
[4]Pierik.R.LM.Gerbera plantlets from in vitro cultivated capitum explant[J].Scientia Hort. 1973,(1):117-120.
[5]Murashige.T.Clonal multiplication of Gerbera through tissue culture[J].Hort.Sci.1974,(9): 175-180.
[6]黄济明.儿种花卉的组织培养[J].植物生理学通讯,1983,(3)：45-46.
[7]黄济明,倪跃之,林满红.非洲菊的快速繁殖[J].园艺学报,1987,14(2)：12-128.
[8]黄玉玲,杨宝明.非洲菊的花托离体培养及快繁技术[J].云南农业科技,1998,(5)：33.
[9]王海琴,冯先桔,李丽,罗君琴.非洲菊的组织培养[J].广西农业科学,2005,36(1)：33-34.
[10]王春彦,高年青,张效平,等.非洲菊幼花托离体培养研究[J].江苏农业科学,200(1)：47-49.
[11]杨波,康明.非洲菊组培快繁技术研究[J].湖北农业科学,2000,(2)：48-49.
[12]郑秀芳,李名扬.非洲菊花托培养和植株再生[J].西南农业大学学报,2001,23(2)：171-173.
[13]雷加容,张跃非,刘兴华,邓晓蓉,余敖.非洲菊的组培快繁技术研究[J].绵阳经济技术高等专科学校学报,2002,19(2)：16-17.
[14]王红梅.非洲菊的组培快繁技术研究[J].甘肃农业科技,2000,(10)：42-43.
[15]席梦利,施季森.非洲菊的离体培养及其快速繁殖[J].南京林业大学学报(自然科学版),2003,27(2)：33-36.
[16]张文珠,林德钦,李梅.非洲菊的快繁技术研究[J].福建农业科技,2002,(1)：17-18.
[17]陈海霞,李茂娟.非洲菊不同发育时期的花托诱导愈伤组织的研究[J].广西农业科学,2008,39(1)：1-5.
[18]杨桂芬,王毓,丁红光.非洲菊组织培养与快速繁殖[J].植物生理学通讯,2000,36(4)：333.
[19]郑秀芳,王桔红,李名扬.影响非洲菊离体培养器官分化的因素[J].江苏林业科技,2002,29(1)：29-31.
[20]鲁雪华,林勇,郭文杰.非洲菊小花托的离体培养[J].亚热带植物通讯,1996,25(2)：21-24.
[21]杨光穗.非洲菊组织培养研究进展[J].热带农业科学,2003,23(1)：57.
[22]刘丽荣,苏荣德,李忠利.非洲菊组织培养繁殖的试验研究[J].辽宁农业职业技术学院学报,2002,4(3)：13-15.
[23]陈发棣,陈滨.非洲菊组织培养中La3+的应用初探[J].园艺学报,2002,29(4)：383-385.
[24]倪丹,杨世湖,徐世清等.非洲菊组培快繁的优化[J].细胞生物学杂志,2002,24 (5)：316-319.
[25]李启任,王云经,夏从龙.非洲菊组培快繁技术研究[J].云南大学学报(自然科学版)1998,20(生物学专辑),560-563
[26]石文山,李树丽,胡敬宜,滕娜.非洲菊的离体培养和快速繁殖技术研究[J].热带农业科学,2006,(12)：44-46.
[27]许彩霞,孙成林,薛炜等.非洲菊组织培养离体快速繁殖[J].内蒙古林业科技,2001,(增刊).
[28]王玉芹,王喜军.非洲菊的组织培养[J]。中国林副特产,2000,(2)：25.
[29]刘福平,林丽仙,邹小鲁等.非洲菊组织培养(简报).亚热带植物通讯,2000,29(2)：52.
[30]陈发棣,李倩中,房伟民等.两个非洲菊品种叶片的离体培养[J].江苏林业科技,1998,25(增刊)：174-176.
[31]曾长立,陶婷,陈禅友.非洲菊的组织培养与快速繁殖[J].江汉大学学报(自然科学版),2005,33(4)：36-38.
[32]戴云新,张健,李敏,李玉娟.不同外植体和激素对非洲菊愈伤组织诱导及芽分化的影响[J].浙江农业科学,2009(4)：695-696.
[33]曹征宇,严志衡,贾明姚愚,顾韵莉,虞冠军.非洲菊离体快速繁殖技术[J].上海交通大学学报(农业科学版),2007,25(4)：395-398.
[34]杨继涛,邹志荣,张素勤,等.植物激素对非洲菊花托愈伤组织诱导的研究[J].西北农业学报,2003,12(3)：133-135.
[35]鲁雪华,郭文杰,林勇.几种因素对非洲菊离体培养再生植株的影响[J].植物生理学通讯,1999,35(5)：372-374.
[36]刘晓燕.非洲菊离体组织培养与快速繁殖[J].耕作与栽培,2001(3)：21.
[37]杨华,阮颖,刘春林等.非洲菊离体再生体系的建立[J].湖南农业科学,2002,(2)：53-54.
[38]徐立,李克烈,李志英等.非洲菊的组织培养和快速繁殖[J]热带农业科学,2000,20(2)：26-27.
[39]张文株,林德钦,李梅.非洲菊的快繁技术[J].福建农业科技,2002(1)：17-18.
[40]王春,余有祥.盆栽非洲菊的组织培养及快速繁殖[J].浙江林业科技,2001,21(3)：30-35
[41]张素勤,邹志荣,耿广东,张亚娟.非洲菊组织培养抑制褐变现象的研究[J].贵州农业科学,2007,35(2)：56.
[42]张仲凯,李毅.云南植物病毒.北京：科学出版社,2001.
[43]除东平,谢联辉.黄瓜花叶病毒亚组研究进展[J].福建农业大学学报,1998,27(1)：82-91.
[44]王健华,王运勤,吉训聪,刘志昕,郑服丛.植物病毒检测技术研究进展[J].热带农业科学,2005,25(3)：71-74.
[45]Derrick K S. Quantitative assay for plant viruses using serologically specific electron microscopy. Virology,1973, (56):652-657.
[46]Louro, D, L esemann D E. Use of protein A-gold complex for specific labeling of antibodies bound to plant viruses-Viral antigens in suspensions. Journal of Virological Methods,1984, (9):107-122.
[47]陈继峰,赵军营,李绍华.果树病毒检测方法及其应用[J].河北林果研究,2005,20(2)：167-173.
[48]陈枝楠,卢高泽,卢俭.PCR应用技术进展简介[J].植物病理学报,1997,27(3)：198-200.
[49]D Colinet, J Kummert, P Lepoivre, et al. Identification of distinct potyviruses in mixedly-infected sweetpotato by the polymerase chain reaction with degenerate primers [J].Phytopathology,1993,84 (1):65-69.
[50]张晶红,徐启江,陈典.唐菖蒲病毒病原鉴定的研究进展[J].黑龙江农学,2008,(6)：159-162.
[51]兰玉菲,郗丽君,张德满等.植物病毒检测技术[J].山东农业科学,2006,5：58-62.
[52]王升吉,尚佑芬,赵玖华等.葡萄卷叶病毒主要检测技术比较[J].山东农业科学,2008,3：95-98.
[53]刘文洪,洪健,陈集双等.百合黄瓜花叶病毒及其检测[J].植物保护,2006,32(1).73-76.
[54]梁巧兰,魏列新,徐秉良.侵染观赏百合的黄瓜花叶病毒血清学检测方法研究[J].甘肃农业大学学报.2009,44(3)：97-101.
[55]Devergne J C,Vardin L,Burckard,J,et al. Compari-song of derect and indirect EL ISA for detecting anti-gencally related cucumovires [J]. Virol method,1981,3 (4):193-199.
[56]杨超,孙秀东.应用RT—PCR检测辣椒上的黄瓜花叶病毒[J].山东农业科学,2009,2：94-96
[57]S. K. Raj,A. Srivastava,G. Chandra,et al. Characterization of cucumber mosaic virus isolate infecting Gladiolus cultivars and comparative evaluation of serological and molecular methods for sensitive diagnosis[J]. Curr. Sci.2002,83:1132-1137.
[58]FANQUETCM, MAYOM A, MAN ILOFF J, et al. Virus Taxonomy-8 th reports of the International Committee on Taxonomy of Viruses [R]. Netherland:Elsevier, Academic Press, 2005.
[59]Brittlebank C C. Tomato diseases. Journal of Agriculture, Victoria 1919,27:231-235.
[60]洪霓.番茄斑萎病毒的检疫技术[J].植物检疫,2006,20(6)：389-392.
[61]Boonham N, P Smith, KWalsh, et al. The detection of Tomato spotted wilt virus (TSWV) in individual thrip s using real time fluorescentRT-PCR (TaqMan). J VirolMethods,2002,101: 37-48.
[62]韩贻仁.分子细胞生物学(第二版)[M].北京：科学出版社,2001.
[63]Ie T S. CMI/AAB Descr PI. Viruses,1970,(39),4pp.
[64]于翠,杨翠云,印丽萍.番茄斑萎病毒--一种值得重视的植物检疫病毒[J].植物检疫,2006增刊,20：47-50.
[65]张仲凯,方琦,丁铭等.侵染烟草的番茄斑萎病毒(TSWV)电镜诊断鉴定[J].电子显微学报,2000,19(3)：339-340.
[661KURSTAK.植物病毒比较诊断指南[M].裴美云译.北京：农业出版社,1993：335-339.
[67]LAWSON R H, D I ENELTM M, HSU H T. The cytopathology of Tospovirus2Host2Plant Interactions [J].Acta Hoticulturae,1996,431:267-290.
[68]Ie T S. CMI/AAB Descr PI. Viruses,1970,(39),4pp.
[69]GonsalvesD, Trujillo E E. Tomato spotted wilt virus in papaya and detection of the virus by EL ISA. Plant Dis.1986,70:501-506.
[70]Wang M, Gonsalves D. EL ISA detection of various tomato spotted wilt virus isolates using specific antisera to structural proteins of the virus. Plant Dis.1990,74,154-158.
[71]Sherwood J L, Sanbom M R, Keyser G C, et al. Use of monoclonal antibodies in detection of tomato spotted wilt virus. Phytopathol,1989,79:61-64.
[72]Hugenot C, Van Den Dobbelsteen, G de Haan P. Detection of tomato spotted wilt virus using mono-clonal antibodies and ribop robes Arch Virol,1990,110,47-62.
[73]郑元仙,刘雅婷.番茄斑萎病毒属病毒检测技术研究进展[J].云南农业大学学报,2009年,24(4),607-613.
[74]BYOUNG-CHEORL K IY, MOLLYM J. Genetics of Plant Virus Resistance [J]. Annual Review of Phytopathology,2005,43:581-621.
[75]RESENDE EW, KITAJ IMA RW, PETERS G D. SerologicalDifferentiation of 20 Isolates of Tomato Spotted wilt Virus [J]. Journal of General Virology,1990,71:2801-2807.
[76]GONSALVESW M D. EL ISA Detection of Various Tomato Spotted wilt Virus Isolates using Specific Antisera to Structural Proteins of the Virus [J]. Plant Disease,1990,74:154-158.
[77]Rice D J, German T L, Mau R F L, et al. Dot blot detection of Tomato spotted wilt virus RNA in p lant and thrip s tissues by cDNA clones. Plant Disease,1990,74:274-276.
[78]Ronco A E,Dal Bo E, Ghiringhelli P D, et al. Cloned cDNA p robes for the detection tomato spotted wilt virus. Phytopathol,1989,79:69-74.
[79]WEEKES R J, MUMFORD R A, BARKER I. Diagnosis of Tospovirus by Reverse-transcription Polymerase Chain Reaction [J]. Acta Hoticultures,1996,431:159-166.
[80]MUMFORD R A, BARKER I, WOOD K R. An Improved Method for the Detection of Tospoviruses Using the polymerase Chain Reaction [J]. Journal of VirologicalMethods, 1996,57:109-115.
[81]CORTEZI J S, WONGJKAEW K S, PEREIRA A M.Identification and Characterization of a Novel Tospovirus Species using a New RT-PCR app roach [J]. Archivesof virology,2001, 146:265-278.
[83]H IGUCH IR, FOCKLER C, DOLL INGER G. Kinetic PCR Analysis:Real2timeMonitoring of DNA Amp lification Reactions [J]. Biotechnology,1993,11:1026.
[82]MORR IS J. Diagnostic Protocols for Regulated Pests Protocoles de Diagnostic Pourles Organismes Reglementes [J]. OEPP/EPPO Bulletin,2004,34 (2):271-279.
[84]CASSI E A, ROBERTSA R G D, L ISA A. Real—timeRT2PCR Fluorescent Detection of Tomato Spotted wilt Virus [J]. Journal of Virological Methods,2000,88:1-8.
[85]付鸣佳,高乔婉,范怀忠.烟草花叶病毒株系鉴定研究进展[J].华南农业大学学报,1997,18(4)：113-117.
[86]张振臣,张丽芳,乔奇,等.河南省地黄病毒病初步鉴定[J].植物病理学报,2004,34(5)：395--399.
[87]王敏,李明福,黄璐琦,等.栽培地黄(Rehmannia glutinosa Liboseh)普遍感染TMV和CMV[J].植物病理学报,2006,36(2)：189-192.
[88]Harrison BD and Nixon HL. Separalon and properties of particles of tobacco rattle virus with different lengths. [J].Gen Microhiol.1958,21:569-581.
[89]Harrison BD and Woods RD. Serotypes and particle dimensions of tobacco rattle viruses from Europe and America.Virology.1966,28:610-620.
[90]马新颖,汪琳,任鲁风,等.应用基因芯片检测烟草脆裂病毒.植物病理学报,2007,37(4)：352-355.
[91]Brunt AA,Crabtree K,Dallwitz MJ,et al.Viruses ofplants. CAB International [M]. Walling ford,UK:1996.
[92]Wellink J,Le Gall O,Sanfacon H,et al.Family Comoviridae.In:Fauquet CM(ed)The Seventh Report of the International Committee on Taxonomy of Viruses[M],Academic Press,San Diego,CA,pp 2000.
[93]Murant A F. Seed and pollen transmission of nematode-borne viruses.Seed Science and Technology,1983,11:973-987.
[94]杜更新,曹新民,李尉民.番茄黑环病毒(TBRV)初步鉴定报告.中国烟草,1992(2)：28.
[95]蔡汉权,李粉玲,林珊珊.花卉脱毒快繁技术研究进展,江西科学,2006,4(24),124-127.
[96]葛胜娟.植物组织培养中的快繁与脱毒技术及其应用[J].中国农学通报,2005,5(21),104-108.
[97]赵军良.植物茎尖培养与无毒种苗生产[J].北方园艺,1995(6)：10-11.
[98]曹为玉,郑燕棠,张福庆等.葡萄茎尖脱毒培养和快速繁殖[J].华北农学报,1993,88(2)：69-72
[99]席梦利,王节萍,章静娟等.宜兴百合脱毒技术[J].江苏农业学报,2001,17(1)：49-51.
[100]张玉清,罗晓芳,田砚亭.葡萄微茎尖培养和葡萄扇叶病毒的ELISA和探针检测[J].北京林业大学学报,1998,20(4)：54-58.
[101]朱德蔚.组织培养与脱毒快繁技术[M].北京：国科学技术出版社,2001。
[102]王永伟,王慧霞,贺丹,何松林.观赏植物病毒病害及病毒脱除研究现状与发展趋势中国农学通报.2008,24(5)：313-317.
[103]谢嘉华,袁建军,花卉脱毒培养与优质种苗生产[J].生物学杂志,2001,18(5)：37-38.
[104]邓衍明,褚芳玲,华如枝.滁菊组织培养脱病毒技术研究[J].安徽农业科学,2007,35(14)：4130,4158.
[105]赵祝成,陈燕贤中国水仙脱毒种苗培育研究取得重大突[J].中国花卉园艺,2003(1)：331.
[106]符国芳,李青.植物组织培养脱毒方法综述.2007,34(3)：255-258.
[107]李文安.业生物工程[M].北京：化学工业出版社,1998：4.
[108]Lizarrage C,Fermandez-Northcote E N1 Detection of potato viruses X and Yin sap extracts by a modified indirect enzyme-linked immunosorbent assay on nitrocellulose membranes (NCM-EL ISA) [J]. Plant Disease,1989,73:11-141
[109]谢嘉华,袁建军.中国水仙(Narcissustazetta varlchinese)的组织培养[J].生物学杂志,2002,19(3)：30.
[110]Engelmann F.Plant cryopreservation:progress and prospects [J]. In Vitro Cellular& Developmental Biology,2004,40:427-433.
[111]蔡汉权,李粉玲,林珊珊.花卉脱毒快繁技术研究进展[J].江西科技,2006(2)：124-127
[112]Helliot B,Panis B,Poumay Y,et al.Cryopreservation for the elimination of cucumber mosaic and banana streak viruses from banana (Musa spp.)[J]. Plant Cell Rep,2002, 20:1117-1122.
[113]高遐红,李梅.提高草莓茎尖组织培养脱毒效果研究[J].中国果树,1994,(2)：5-6.
[114]周厚成,何水涛.草莓病毒病研究进展[J].果树学报,2003,(5)：421-426.
[115]高庆玉,李光裕,周恩.关于草莓脱毒技术的研究[J].东北农学院学报,1993,(3)：231-23.
[116]Thomson A J, et al.The evaluation of potato somaclones. In:Semal Jed. Somaclonal Variations and Crop Improvement. Martnus Nijhoff Publishers. Dordrecht.1986:236-243.
[117]孔宝华,蔡红,陈海如等.花卉病毒病及防治[M].北京：中国农业出版社,2002：119-121.
[118]Sanchez Navarro J A. Comparative analysis of ELISA, nonradioactive molecular hybridization and PCR for the detection of Prunus necrotic ring spot virus in herbaceous and prunus host[J]. Plant Pathol,1998,47:7802786.
[119]Sanchez Navarro J A. Simultaneous detection and identificationof eight stone fruit viruses by one-step RT-PCR,European[J] Journal of Plant Pathology,2005,111:77284.
[120]兰平,李文风等.热处理结合茎尖培养去除甘薯丛枝病植原体[J].西北农林科技大学学 报,2001,6：1-4.
[121]F.A.Hakkaart,G.Hartel.Virus eradication from some Pelargonium zonale cultivars by meristem-tipculture[J]. European Journal of Plant Pathology,1979,85:3-46
[122]R.Ram,N.Verma.Indexing and production of virus-free chrysanthemums.Biologia plantarum,2005,49(1):149-152
[123]G.A.Manganaris,A.S.Economou,et al.Elimination of PPV and PNRSV through thermotherapyand meristem-tip culture in nectarine.Plant Cell Rep,2003,22:195-200.