柑橘茎尖微芽嫁接脱毒及黄龙病和衰退病分子检测的研究
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摘要
本实验采集带有典型衰退病症状的江西新余蜜柑母株茎尖进行了茎尖微芽嫁接(STG),获得了成活苗169株;并对稻叶蜜柑、市文蜜柑、翡翠柚、处红柚、瓯柑2003年以来已获得的186株STG苗及母株进行了黄龙病和衰退病分子检测;对不同浓度病毒唑对茎尖微芽嫁接成活率的影响进行了研究,主要的结果如下:
     1.以带典型黄龙病症状并经分子检测的柑橘作正对照,对即将出圃的1-3年龄STG苗186株进行了PCR检测,被检测样本均没有检测到黄龙病病原,无毒率100%。
     2.对瓯柑、稻叶蜜柑、市文蜜柑、翡翠柚、处红柚等6个品种的母株和茎尖嫁接苗进行了RT-PCR检测,结果所有样品的母株均扩增出了衰退病特异性条带,而STG嫁接成活苗的无毒率最高仅为61.1%,证明茎尖嫁接方法并不能完全脱除衰退病病原。
     3.对比了不同浓度病毒唑对嫁接成活率的影响,初步筛选出了适宜嫁接的最适浓度为20mg/L。
In this study, the shoot-tips of Citrus sutsuma Marc from Xinyu city of Jingxi province were used as explants to perform shoot-tip grafting (STG) , 169 STG plantlets were obtained; all the mother plants of Citrus sutsuma Marc cv Daoye、Citrus sutsuma Marc cv Shiwen、Citrus grandis osback cv Feicui、Citrus grandis osback cv Chuhong、Citrus Suavissima Hort ex Tanaka and 186 STG plants ever since 2003 have been determinated with PCR and RT-PCR. We evaluated the existence of pathogery of BLO and CTV. In addition, we studied the effect of Virazole treatment on the shoot-tip grafting survival rate, the main results are as following:
     1. 186 STG plants of 1-3 years old were identified by PCR technique, BLO pathogen was not found, the virus-free rate was 100%.
     2. We detected the five varieties citrus mother plants and STG plants with RT-PCR, as a result, all of the mother plants could amplified Tristeza bands, but the highest virus-free rate of grafting plants only was 61.1%, and the results may prove that not all shoot-tip grafting can remove the CTV completely.
     3. Contrasted the effect of different concentration virazole on grafting survival rate, and the optimum of viratole is 20mg/L.
引文
1.陈青瑛,陈景耀.龙眼茎尖离体培养及其脱毒效果.植物生理学通讯,1996,32(2):126-131
    2.陈雄庭,Chee Kheng Hoy,郑学勤等.柚木离体快速繁殖技术.热带作物学报,2001,22(3):59-63
    3.陈杰忠,陈全友,吴云伦.不同柑橘品种衰退病—茎陷点症状调查.中国柑橘,1992,21(2):26-28
    4.陈泽雄.利用柑橘茎段诱导不定芽改进微芽嫁接脱毒技术的研究.[硕士学位论文].武汉:华中农业大学图书馆,2005
    5.陈国庆,王宏祥,严申祥.构头橙种子中柑橘衰退—茎陷点病毒的检测.植物病毒学报,1997,27(1):75-78
    6.邓小玲,唐伟文.应用PCR技术检测柑橘黄龙病病原的研究.华南农业大学学报,1996,17(3):119-120
    7.窦道龙,王冰山,唐益雄.棉花高质量总RNA提取的一种有效方法.作物学报,2003,29(3):478-479
    8.郭文武,邓秀新.柑橘黄龙病及其抗性育种研究(综述).农业生物技术学报,1998,16(1):37-41
    9.谷守芹,解灵君,范永山.植物组织总RNA提取的常用方法及优化策略.保定师范专科学校学报,2005,18(2):2-4
    10.何天富.柑橘学.北京:中国农业出版社,1999,185-197
    11.何新华,蒋元晖,赵学源等.应用热处理—茎尖嫁接方法培育无碎叶病毒的柑橘.果树科学,1993,10(2):95-96
    12.何新华,蒋元晖,赵学源.柑橘碎叶病毒的脱毒.云南农业大学学报,1995,10(2):100-101
    13.侯义龙,张开春,吴禄平.果树组织中总RNA提取的新方法.沈阳农业大学学报,2002,33(2):122-124
    14.洪霓,王国平,张尊平.梨病毒脱除技术研究.中国果树,1995,4:5-7
    15.黄萱,高丽美,张永彦.一种优化的植物总DNA提取方法.西北植物学报,2004,24(6):1103-1106
    16.黄训才.湖南主要柑橘病毒病的分子检测及其毒系分型研究.[硕士学位论文].长沙:湖南农业大学,2006
    17.黄治远,漆巨贤等.柑橘茎尖嫁接脱毒苗培育技术初探.四川果树,1996,2:10-11
    18.姜玲,万蜀渊.提高柑橘茎尖微芽嫁接成活率的研究.湖北农业科学,1992,4:26-28
    19.姜玲,万蜀渊,王映红.柑橘茎尖嫁接操作方法的改进及研究.华中农业大学学报,1995,14(4):381-385
    20.纪彦.樱桃脱病毒及病毒检测技术研究.[硕士学位论文].长春:辽宁师范大学,2002
    21.蒋元晖,赵学源,苏维芳等.通过茎尖嫁接脱除柑桔黄龙病病原体.植物保护学报,1987,14(3):184,162
    22.柯冲.应用抗生素治疗柑橘黄龙病的研究.福建农科院学报,1988,3(1):1-10
    23.李红叶,陈力耕,周雪平.柑橘病毒与类似病毒病分子生物学和抗病毒基因工程研究进展.果树科学,2000,17(2):131-137
    24.李师翁.苹果茎尖培养和脱毒苗快速繁殖研究.甘肃农业科技,1994,6:17-19
    25.林尤剑,谢联辉,Powel CA.橘蚜传播柑橘衰退病毒的研究进展.福建农业大学学报,2001,30(1):59-66
    26.刘柏玲.柑橘黄龙病的茎尖微芽嫁接脱毒及PCR检测的研究.[硕士学位论文].武汉:华中农业大学图书馆,2004
    27.刘建雄,舒广平.柑桔茎尖嫁接脱除裂皮病技术研究.湖南农业科学,1988,3:30-32
    28.马凤桐.柑橘属茎尖嫁接脱毒的研究.植物学报,1989,31 (7):565-568
    29.宋瑞琳,柯冲.在试管中草尖嫁接脱除柑枯黄龙病病原的研究.福建省农科院学报,1987,5(1):20-26
    30.宋瑞琳,吴如健,柯冲.茎尖嫁接脱除柑橘主要病原的研究.植物病理学报,1999,29(3):275-279
    31.宋震.柚类柑橘衰退病毒CTV强弱毒分离株的分子鉴定.[硕士学位论文].重庆:中国农业科学院柑橘研究所,2005
    32.谭荣荣.物理及化学处理脱除砂梨潜隐病毒研究.[硕士学位论文].武汉:华中农业大学图书馆,2006
    33.田亚南,柯穗,柯冲.柑橘黄龙病诊断法的研究进展.福建农业学报,1998,13(1):27-35
    34.田亚南,柯穗,柯冲.应用聚合酶链式反应(PCR)技术检测和定量分析柑橘黄龙病病原.植物病理学报,1996,26(3):243-250
    35.王文锋,肖月华,侯磊.棉花总RNA的快速提取方法.河南农业大学学报,2002,36(3):229-232
    36.王子成.柑橘种质资源离体保存研究.[博士研究生学位论文].武汉:华中农业大学图书馆,2002
    37.王子成,邓秀新.柑橘体细胞杂种超低温保存厚的植株再生.园艺学报,2004,31(2):215-216
    38.王景雪,孙毅.一种简便实用的植物总DNA提取方法.山西大学学报,2000,23(3):271-272,
    39.王春梅.湖南柑橘黄龙病检测及DNA片段克隆.[硕士学位论文].长沙:湖南农业大学,2006
    40.吴力游,刘学端,萧启明.柑橘茎尖脱毒技术的改进.湖南农学院学报,1992,18:161-166
    41.吴力游,萧启明,刘学端等.湖南省柑橘良种的脱毒与检测的研究.湖南省农学院学报, 1993,19(3):240-247
    42.吴文,马培恰,唐小浪等.柑橘无病毒树的生长和结果表现初报,中国南方果树,2002,31(5):3-4
    43.吴世盘,李剑书.柑橘黄龙病和斑黄病病原体的初步观察.中国农业科学,1986,1:73-75
    44.许宏冠.几种果树的脱毒方法.植物检疫,1989,3:5-6
    45.许智宏.植物生物技术.上海:上海科学技术出版社,1997,118-150
    46.徐昌杰,陈昆松,张波.柑橘组织RNA提取方法研究.果树学报,2004,21(2):136-140
    47.薛光荣,杨振英,洪霓.茎尖培养等处理脱除梨病毒的技术研究.中国果树,1996,3:9-11
    48.余东东.柑橘衰退病的快速检测技术研究[硕士学位论文].武汉:华中农业大学图书馆,2004
    49.杨振德.成年型桂夏橙茎段组织培养中的脱落现象及其生理原因初探.广西农业大学学报,1992,11(4):38-42
    50.乙引,罗在柒,柯波.几种DNA提取方法的比较.生物学通报,2004,39(3):55
    51.赵双宜,吴耀荣,夏光敏.介绍一种简单高效的植物总RNA提取方法.遗传,2002,24(3):337-338
    52.赵学源,蒋元晖,李世菱等.我国柑桔栽培品种的裂皮病鉴定和脱除.园艺学报,1986,13(2):91-94
    53.张年辉,韦振泉,何军贤.一种高效经济的高质量植物RNA提取方法.生物化学与生物物理进展,2004,31(10):947-953
    54.张博,张露,诸葛强.一种高效的树木总DNA提取方法.南京林业大学学报,2004,28(1):13-17
    55.张志忠,吴菁华,吕柳新.一种适于PCR检测的DNA微量提取方法.亚热带植物科学,2004,33(1):29-30
    56.张宁,王风山.DNA提取方法进展.中国海洋药物,2004,2:34-36
    57.张才建,王少成.柑橘无病毒苗木繁育技术及应用推广建议.西南园艺,2000,28(3):25
    58.周常勇.我国柑橘衰退病的发生概况与展望.第一次植物病毒与病毒病防治学术讨论会论文集,北京:中国农业科技出版社,1997,182-187
    59.张秋胜,舒广平,宋顺华.柑橘茎尖嫁接技术的改进.中国柑橘,1991,20(2):11-13
    60. Avijit Roy, Amer Fayad, G. Barthe. A multiplex polymerase chain reaction method for reliable, sensitive and simultaneous detection of multiple viruses in citrus trees. Journal of Virological Methods, 2005, 129: 47-55
    61. Avijit Roy, R. H. Brlansky. Genotype classification and molecular evidence for the presence of mixed infections in Indian Citrus tristeza virus isolates. Arch Virol, 2004
    62. Roy, P. Ramachandranl, R. H. Brlansky. Grouping and comparison of Indian citrus tristeza virus isolates based on coat protein gene sequences and restriction analysis patterns. Arch Virol, 2003, 148: 707-722
    63. Bar-Joseph M, Marcus R, Lee RF. The continuous challenge of citrus tristeza virus control. Annu Rev Phytopathol, 1989,27: 291-316
    64. Deng X, He W, Tang W. Diagnosis and detection of citrus Huanglongbing in infected trees without any symptoms by Nested PCR. 1998, XIV Conference of IOCV Programme&Abstracts: 65.Campinas, S P Brazil
    65. Febres V, Niblett C, Lee R. Characterization of grapefruit plants transformed with citrus tristeza closterovirus genes. Plant Cell Reports, 2003,21(5): 421-428
    66. Gede SUASTIKA, Tomohide NATSUAK. Nucleotide Sequence of Citrus Tristeza Virus Seedling Yellows Isolate. Plant Pathol, 2001, 67: 73-77
    67. Gamier M, Bove JM. The greening organism is a gram negative bacterium In: Garnsev SM. Timmer LW. Dodds JA (eds). Proc 9th Conf Int Org, Citrus Virol. Riverside, 1984: 115-124
    68. Gamier M, Bove JM. Transmission of the organism associated with citrus greening diseases from sweet orange to periwinkle by dodder. Phytopathology, 1983, 73: 1358-1363
    69. Gamier M. Distribution of greening liberobacteria species in fifteen African and Asian countries Pro(abstracts) 13th Conf IOCV, 1995: 103
    70. Gamier M, Gao SJ, He YL. Study of the greening organism (GO) with monoclonal antibodies: serological identification morphology serotypes and purification of the GO. In: Weathers LCx Cohen M(eds) Proc 11th Conf Int Org Citrus Virol. Riverside, 1991: 428-435
    71. Gibbons N E, Murray R G E. Proposals concerning the higher taxa of bacteria. Inter Jour Syst. Bacteriol, 1978,28: 1-6
    72. Jagoueix S, Bove JM, Gamier M. The phloem-limited bacterium of greening disease of citrus is a member of a subdivision of the proteobacteria. Int J Syst Bacteril, 1994,44(3): 379-386
    73. Jagoueix species S, Bove J M, Gamier M. PCR detection of the Candidates liberobacter disease of citrus. Molecular and Cellular probes, 1996, 10: 43-50
    74. Kobayashi S, Uchimiya H, Expression and integration of a foreign gene in orange (Citrus siencsis Osb.) protoplasts by direct DNA transfer. Jpn J Genet, 1989, 64: 91-97
    75. Kawai A, et al. Detection of citrus tatter leaf virus by enzyme-linked immunosorbent assay (ELISA), Annals of the Phytopathological Society of Japan, 1990, 56: 342-345
    76. LIU Yong-zhong, LIU Qing, TAO Neng-guo DENG Xiu-xin. Efficient Isolation of RNA from Fruit Peel and Pulp of Ripening Navel Orange (Citrus siueusis Osbeck). Journal of Huazhong Agricultural University, 2006, 3: 300-304
    77. Mukhoropagation S, Jaishree kai, Sharma BC. Micropropagation of Darjeeling orange by shoot-tip-grafting. Journal of Hortscience.1997, 72(3): 493-499
    78. Murashinge T, Bitters P, Rangan TS. A technique of shoot-apex grafting and its utilization towards recovering virus-free citrus clones. Hortscience, 1972, 7: 118-119
    79. M urashige T, Bitters P W, Rangan M E et al. A technique of shoot apex grafting and its utilization towards recovering virus-free citrus clones. Hortiscience 1972, 7: 118-119
    80. Navarro L, Roistacher N C, Mnrshige T. Improvement of shoot-tip grafting in vitro for virus-free citrus. J.Amer. Soc. Hort. Sci., 1975, 100 (5): 471-479
    81. Navarro L, Jucureza J, Ballester J F. Elimination of some citrus pathogens producing psorosis-like leaf symptoms by shoot-tip-grafting in vitro. Riverside In Proc 8th Conf IOCV, 1980: 162-166
    82. Navarro L. Citrus shoot-tip grafting in vitro (STG) and its applications: a review Proc. Int. Soc. Citriculture, 1981, 1: 452-456
    83. Robacker CD, Chang CJ. Shoot-tip culture of Muscadine grape to eliminate pierces disease bacteria. Hortscience, 1992, 27(5): 449-450
    84. Sambade, L. Rubio, R. Flores. Polymorphism of a specific region in gene p23 of Citrus tristeza virus allows discrimination between mild and severe isolates. Arch Virol, 2003, 148: 2325-2340
    85. Salvatore Davino, Luis Rubioand, Mario Davino. Molecular analysis suggests that recent Citrus tristeza virus outbreaks in Italy were originated by at least two independent introductions. European Journal of Plant Pathology, 2005, 111: 289-293
    86. Villechanoux S, Gamier M, Renaudin J. Detection of several strains of the bacteria-like organism of citrus greening disease by DNA Probes. Curr Microbiol, 1992,24: 89-95 .
    87. Whiteside J, Garmsey SM, Timmer LW. Compendium of citrus diseases. St. Paul: American Phytopathology Society Press, 1988,48-50

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