植物组培污染防治的研究
|
摘要
本试验于2000～2002年在山东农业大学和泰安山口格林农业生物技术有限公司进行。试验试材主要有果树(樱桃colt砧木、草莓、冬枣、石榴)、花卉(月季、一品红、蔷薇)、林木(八里庄杨、速生杨、日本构树)。针对当前植物组织培养中污染率过高，在科研和生产中造成人力、物力和财力巨大浪费的现状，对植物组织培养污染进行了系统的研究。主要研究结果如下：
1．根据微生物的菌落特征、光学显微镜观察、昆虫的形态特征和生活习性进行了污染物种类的鉴定，认为污染物主要有真菌(青霉菌、镰刀菌、黑曲霉)、细菌(革兰氏阳性和革兰氏阴性)和昆虫(螨和蓟马)。
2．在植物组织培养条件下，以菌落直径为指标，测定了不同真菌的繁殖生长进程。利用分光光度计采用比浊法测定了细菌的繁殖生长状况。从真菌和细菌的生长曲线可以看出，两者都有一个快速生长阶段。
3．对培养瓶和外植体各部位的菌类分布，做了系统调查，试验结果表明：外植体各部位的菌类分布有一定的差异，新叶和芽带菌较少；培养瓶的瓶壁和封口纸都能携带污染菌，成为污染源之一。
4．对培养室和接种室采用紫外线和臭氧发生器两种灭菌方法进行灭菌比较试验，认为臭氧比紫外灯方式杀菌能力强，消毒效果好。
5．试验证明植物导管直径大于细菌100倍以上，细菌完全能够通过外植体导管进入到外植体内部，成为外植体的内生菌，引起接种后的组培苗污染。
6．提出植物组培苗污染防治的四项措施：第一，在温室中采的外植体接种成活率高于大田中采的外植体，同时对大田中的外植体亲本进行预处理也能提高接种成活率。第二，对组培苗要勤检查，及时挖除污染培养基和转移未污染的外植体。第三，在培养基中加入防腐剂以及抗生素和医用杀菌剂混合物，进行综合防治。第四，已污染的组培苗采用滤纸桥瓶外生根法挽救。
Studies on the contamination of plant tissue culture was studied in Shandong Agricultural University and Tai'an Agricultural Science and Technology Company during 2000-2002,using some kinds of fruit trees (P sernlata Ledeb; C erasiis vulgavis MilL Fradaria vesca L^ Ziziphus jujube mill; Z saliva Gaevtn^ Punica granat um L.),flowes (Rosa rugosa^ Euphorbia pulcherrima^ R.multiflora) and forest trees (Balizhuang-yang^ Populus L. -. Broussonetiapapyrifera L.) as materials. The main results were as follows:
1. According to colony character of the microbe, taking photographs by electron microscope, shape character, habit and characteristics of live of insect , the identification is made. As is thought that contamination was caused mainly by fungus (Fusarium > Aspergillus niger^ Penicillium ).bacterium(G^ and G~ ) and insect (acarus and thrips ).
2. Under the condition of plant tissue culture, having the diameter of fungus community as guide line, the propagation and growth of different fungus is measured. Status of propagation and growth of bacterial is measured by spectrophotometer. From the growth status of fungus and bacteria, a rapid growth phase could be seen in both of them.
3. The distributing of fungoid both explant and culture bottle was investigated in system, the result of the experiment showed that there was some difference in the distributing of fungoid between every part of explant, and there was less fungoid in the newly born leaves and buds; The wall of the culture bottle and the seal paper could also carry fungoid which brought about contamination of plant tissue culture, and both of them became one of the fountainhead of contamination.
4. The fungoid on the culture room and inoculation room was extinguished by using two kinds of method including ultraviolet radiation and ozonizer. As was thought that it was better to extinguish the fungoid using ozonizer than ultraviolet radiation, the effect of ozonizer was better and it could thoroughly extinguish the fungoid without leaving any dead angle.
5. When the diameter of the plant trachea was 100 times bigger than that of bacteria,
the experiment showed that bacterial could thoroughly enter into the inside of the explant from the outside of the explant and turned into the endogenesis fungus, then that caused the contamination of the plantlet after the inoculation.
6. Four steps was summed up on the prevention and cure about the contamination of plant tissue culture: First, the material used as inoculation should be collected in spring and autumn when the propagation of fungoid was slow; sprouting inside the room by collecting rigid branch outside the room in winter, then inoculating with young buds; avoiding as possible inoculation in summer, and spraying a certain concentration antiseptic medicine before collecting the material from the female parent. Second, check-up of the plantlet should be made frequently, and the contaminated medium should be excavated and the explant uncontaminated should be transferred in time. Third, integration of prevention and cure could be made by adding antibiotic and antiseptic used in medicine; Fourth, the plantlet having been contaminated could be saved by rooting in filter-paper bridge outside the bottle.
1．根据微生物的菌落特征、光学显微镜观察、昆虫的形态特征和生活习性进行了污染物种类的鉴定，认为污染物主要有真菌(青霉菌、镰刀菌、黑曲霉)、细菌(革兰氏阳性和革兰氏阴性)和昆虫(螨和蓟马)。
2．在植物组织培养条件下，以菌落直径为指标，测定了不同真菌的繁殖生长进程。利用分光光度计采用比浊法测定了细菌的繁殖生长状况。从真菌和细菌的生长曲线可以看出，两者都有一个快速生长阶段。
3．对培养瓶和外植体各部位的菌类分布，做了系统调查，试验结果表明：外植体各部位的菌类分布有一定的差异，新叶和芽带菌较少；培养瓶的瓶壁和封口纸都能携带污染菌，成为污染源之一。
4．对培养室和接种室采用紫外线和臭氧发生器两种灭菌方法进行灭菌比较试验，认为臭氧比紫外灯方式杀菌能力强，消毒效果好。
5．试验证明植物导管直径大于细菌100倍以上，细菌完全能够通过外植体导管进入到外植体内部，成为外植体的内生菌，引起接种后的组培苗污染。
6．提出植物组培苗污染防治的四项措施：第一，在温室中采的外植体接种成活率高于大田中采的外植体，同时对大田中的外植体亲本进行预处理也能提高接种成活率。第二，对组培苗要勤检查，及时挖除污染培养基和转移未污染的外植体。第三，在培养基中加入防腐剂以及抗生素和医用杀菌剂混合物，进行综合防治。第四，已污染的组培苗采用滤纸桥瓶外生根法挽救。
Studies on the contamination of plant tissue culture was studied in Shandong Agricultural University and Tai'an Agricultural Science and Technology Company during 2000-2002,using some kinds of fruit trees (P sernlata Ledeb; C erasiis vulgavis MilL Fradaria vesca L^ Ziziphus jujube mill; Z saliva Gaevtn^ Punica granat um L.),flowes (Rosa rugosa^ Euphorbia pulcherrima^ R.multiflora) and forest trees (Balizhuang-yang^ Populus L. -. Broussonetiapapyrifera L.) as materials. The main results were as follows:
1. According to colony character of the microbe, taking photographs by electron microscope, shape character, habit and characteristics of live of insect , the identification is made. As is thought that contamination was caused mainly by fungus (Fusarium > Aspergillus niger^ Penicillium ).bacterium(G^ and G~ ) and insect (acarus and thrips ).
2. Under the condition of plant tissue culture, having the diameter of fungus community as guide line, the propagation and growth of different fungus is measured. Status of propagation and growth of bacterial is measured by spectrophotometer. From the growth status of fungus and bacteria, a rapid growth phase could be seen in both of them.
3. The distributing of fungoid both explant and culture bottle was investigated in system, the result of the experiment showed that there was some difference in the distributing of fungoid between every part of explant, and there was less fungoid in the newly born leaves and buds; The wall of the culture bottle and the seal paper could also carry fungoid which brought about contamination of plant tissue culture, and both of them became one of the fountainhead of contamination.
4. The fungoid on the culture room and inoculation room was extinguished by using two kinds of method including ultraviolet radiation and ozonizer. As was thought that it was better to extinguish the fungoid using ozonizer than ultraviolet radiation, the effect of ozonizer was better and it could thoroughly extinguish the fungoid without leaving any dead angle.
5. When the diameter of the plant trachea was 100 times bigger than that of bacteria,
the experiment showed that bacterial could thoroughly enter into the inside of the explant from the outside of the explant and turned into the endogenesis fungus, then that caused the contamination of the plantlet after the inoculation.
6. Four steps was summed up on the prevention and cure about the contamination of plant tissue culture: First, the material used as inoculation should be collected in spring and autumn when the propagation of fungoid was slow; sprouting inside the room by collecting rigid branch outside the room in winter, then inoculating with young buds; avoiding as possible inoculation in summer, and spraying a certain concentration antiseptic medicine before collecting the material from the female parent. Second, check-up of the plantlet should be made frequently, and the contaminated medium should be excavated and the explant uncontaminated should be transferred in time. Third, integration of prevention and cure could be made by adding antibiotic and antiseptic used in medicine; Fourth, the plantlet having been contaminated could be saved by rooting in filter-paper bridge outside the bottle.
引文
[1] 李春燕．组织培养中青霉素对细菌污染的抑制作用，东北林业大学学报，2000，28(5)：97—98
[2] 朱广廉．植物组织培养中灭菌和无菌操作的几个问题，植物生理学通讯，1995，31(5)：373-375
[3] 朱广廉．植物组织培养中的外植体灭菌．植物生理学通讯，1996，32(6)：444-449
[4] 林盛，马崇烈．组织培养中污染的控制．广西农业科学，1994(2)：87-88
[5] 何云芳，余有祥．金线菊与菊芋在组织培养中细菌污染的防治，浙江林业科技，1997(4)：18-22
[6] 王玉军．山东农业大学硕士研究生毕业论文，2000。
[7] 杨红花．山东农业大学硕士研究生毕业论文，2001。
[8] 王心钗编．植物显微技术[M]，1986，福建教育出版社。
[9] 邹文欣，谭仁祥．植物内生菌研究新进展，植物学报，2001，43(9)：881-892
[10] 韩美丽，陆荣生等．绿巨人组培继代过程中玻璃化苗及细菌污染的消除方法研究，广西林业科学，1999，28(1)：16-19
[11] 蔡静，王亚静等．O_3消毒机与紫外线消毒比较，湖北医科大学学报，1999，20(3)：263-264
[12] 韩玉坤，周华等．臭氧与紫外线对医院病房空气消毒效果比较，中国消毒学杂志，1997，14(3)：185
[13] 奏启玲，贾瑞萍等．医院内三种空气消毒方法比较，齐鲁护理杂志，1999，5(3)
[14] 波边一功，陈微微．抗真菌抗生素，国外医药：抗生素分册，1989，10(5)：377-379
[15] 翟建中，顾梅俏．长春蔓组培生产中污染菌的防除，森林病虫通讯，1999，4：30-32
[16] 肖显华，王顺珍等．植物材料表面消毒方法的改进，生物技术，1999，9(1)：43-45
[17] 黄惜等．抗生素在防止香蕉组培苗污染上的应用研究，海南岛农业科技，1995，(2)：18-20
[18] 谭文澄，戴策刚．观赏植物组织培养技术，1997。
[19] 井上真由美．微生物灾害及其防止技术，上海科学技术出版社。
[20] 邵力平，沈瑞祥等．真菌分类学，中国林业出版社。
[21] 魏景超．真菌鉴定手册，上海科学技术出版社。
[22] 山东农业大学，莱阳农学院编，微生物学试验，北京农业大学出版社。
[23] 曹孜义等．编实用植物组培培养技术教程，1996，甘肃科学技术出版社。
[24] 沈惠娟．木本植物组织培养技术，1992，中国农业科学出版社。
[25] 张开旺．抗生素对大豆组织培养物的抑制效应，1991(1)：45-47
[26] 杨颐康．微生物学，1987，高等教育出版社。
[27] 陈正华主编．木本植物组织培养及其应用，1986，高等教育出版社。
[28] 许婉芳，龚福生等．杀菌剂对金线莲组织培养中微生物污染的抑制作用，福建果树，1999(110)：6-7
[29] 张贯新，晋萍．臭氧用于空气消毒效果观察，中原医刊，1999，26(6)：32-33
[30]陈华癸、樊庆笙主编的《微生物学》，1991
[31]任丽霞，秦香丽，浅谈手术室交叉感染的预防和控制，山西临床医药杂志，1999 6(3)：211-212
[32]Boxus, P.H.& Terzi, J.-M. Control of accidental contamination during mass propagation. Acta Horticulturae, 1988,225:189-193
[33]Blake, J. Mites and thrips as bacterial and fungi vectors between plant tissue cultures. Acta Hortuculturae ,1988, 225:163-166
[34]Boxus,Ph. And Terzi, J.M.,1987. Big losses due to bacterial contaminations can be avoided in mass propagation scheme. Acta Hortic.212(1): 91-93
[35]Bhansali, R.R. and Singh, k., 1986. In vitro emergence of multiple plantlets from smut infected apical tips of sugareane nodal buds. Indian Phytopathol, 39:582-583.
[36]Brenner, D.J.,1984 Description of the species in the family Enterobacteriaceae. In Bergey's Manual of Systematic Bacteriology ed. Krieg,N.R.&Holt, J.G.pp.408-420. Baltimore:Williams and Wilkins.
[37]Bastiaens, L., 1983. Endogenous bacteria in plants and their implications in tissue culture. A review Med. Fac. Landbourw. Rijksuniv. Gent. 48/1:1-11.
[38]Constantine, D. R., 1986. Micropropagation in the commercial environment. In: Plant Tissue Culture and Agricultural Applications (eds. L. A. Withers and P.G. Alderson). Butterworth, London. Journal-of-Agriculture-and-Forestry. 1999, 48: 4, 79-91
[39]Chen-ChiaChung; Huang-ChingWen; Chen-CC; Huang-CW Cotty,-P.J. Method for the control or prevention of aflatoxin contamination using a non-toxigenic strain of Aspergillus niger flavus. United states Department of Agriculture patents. [Washington, D.C.?:The Department, 1900?-.Mar15,1994.(5,294,442) 1 p.88-92
[40]C. Lelfert. et al. Eilmation of Lactobacillus plantarum,Corynebacterium spp.,Staphylococcus saprophyticus and Pseudomonas paucimobills from micropropagated Hemerocallis,Cholsya and Delphinium cultures using antibiotics. Jurnal of Applied Bacteriology, 1991,71:307—330
[41]Casseus.A.c problem in tissue culture : culture contamination in micropropagation technology and application[M]. Kluwer. Academic publishers. 1991, 31-34
[42]Cornu, D.& Michel, M.F. Bacteria contamination in shoot cultures of Prunus avium L. choice and phytotoxicity of antibiotics. Acta
Horticulurae ,1987,212:83-86
[43] Constantine, D. R. , 1986． Micropropagation in the commercial environment. In: Plant Tissue Culture and its Agricultural Applications (eds., L. A. Withers and P. G. Alderson). ISBN 0-407-00921-3．
[44] Cassells, A.C. and Long, R.D., 1982． The elimination of potato viruses X,Y,S and M in meristem and explant cultures of potato in the presence of Virazole. Potato Res. 25: 165-173
[45] de Fossard, R.A. and Watanabe.I., 1988． Development of a photoautotrophic tissue culture system for shoots and /or plantlets at rooting and acclimatization stages. Acta Horticulturae 230:153-158
[46] Dahiya, J.S., Woods, D.L., and Tewari, J. P., 1988． Control of Rhizoctonia solani, causal agent of brown girding root rot of rapeseed, by Pseudomonas fluorescens. Bot. Bull. Acad. Sinica 29: 135-142．
[47] Davison, J., 1988． Plant beneficial bacteria. Biotechnology 6:282-286．
[48] Debergh, P.C.& Vandershaeghe, A.M. Some symptoms indicating the presence of bacterial contaminations in plant tissue cultures. Acta Horticulturae, 1988, 225:77-83
[49] Debergh, P. and Maene,L., 1984． Pathological and physiological problems related to the in vitro culture of plants. Parasitica 40/2-3:69-75．
[50] Dychdala, G.R., 1983． Chlorine and chlorine compounds. In: Disinfection, Sterilization and Preservation. 3rd Edition (ed. S.S. Block). Lea and Febiger, Philadelphia: 157-182．
[51] Edvvin B. Herman, Non-axenic plant tissue culture: possibilities and opportunities. Acta horticulturae, 1990, 280:1990．
[52] Enjalric,F.,Carron, M.P.& Lardet, L. Contamination of primary cultures in tropical areas: The case of Hevea brasiliensis. Acta Horticulturae ,1988,225:57-65
[53] Enjalric,F..1986． Le probleme des infections en zone subtropicale humide. Rapport IRCA/ClRAD3/86:14p.
[54] Falkiner,-F.R. Antibiotics in plant tissue culture and micropropagation-what are we aiming at. Pathogen and microbial contamination management in micropropagation/Dordrecht; Boston:Kluwer Academic Publishers, c1997． ,155-160
[55] Falkiner, F.R Strategy for the selection of antibiotics for use against common bacterial pathogens and endophytes of plants. Acta Horticulturae., 1988,225:53-57
[56] Fisse,J., Batalle, A. & Pera, J. Endogenous bacteria elimination in ornamental plants. Acta Horticulturae, 1987, 212: 87-90
[57] Fisse, J.. Batlle, A. and Pera, J., 1985． Endogenous bacteria elimination in ornamental explants. Symposium: In vitro problems related to mass propagation horticultural plants. Gembloux 16-20 Stember 1985．
[58] Holford.-P.;Newbury,-H.J. The effects of antibiotics and their breakdown products on
the in vitro growth of Antirrhinum majus. Plant-Cell-Rep. Berlin, W. Ger.: Springer International. 1992． V.11(2) p.93-96
[59] Herman, E.B., Toward control of micropagation contamination. Agericell Report, 19879:33-35
[60] Herman, E.B., 1987． Toward control of micropropagation contamination. Agricell Report 9: 33-35．
[61] Juhnke, M.E., Mathre, D.E., and Sands, D.C., 1987． Identification and characterization of rhizosphere-competent bacteria of wheat. Appl. Environ. Microbiol.53:2793-2799．
[62] Jong,S.C., 1987． Prevention and control of mite infestations in fungus cultures. American Type Culture Collection (ATCC) Quarterly Newsletter 7(1) : 1 and 7．
[63] Kelman, A. & Dickey, R.S. 1980 Soft rot or 'carotophora'group. In Identification of Plant Pathogenic Bacteria ed. Schaad, N.W. pp.31-35． Paul: American Phytopathological Society.
[64] Langens-Gerrits,-M; Albers,-M.; Klerk,-G.J.D. Hot-water treatment before tissue culture reduces initial contamination in Lilium and Acer. Plant-cell,-tissue-organ-cult. Dordrecht, The Nether lands: Kluwer Academic Publishers. 1998． v.52( 1/2) p. 83-88
[65] Leggatt, I., Waites, W. M. Leifert, C. & Nicholas, J. R. (1988) Characterisation of micro-organisms isolated from plants during micropropagation. Acta Horticulturae, 1989a, 225: 93-102 Kluwer Academic Publishers. 1998． v.52(1/2) p.83-88
[66] Long,-R.D. Photoautotrophic micropropagation-a strategy for contamination control. Pathogen and microbial contamination management in micropropagation/Dordrecht;Boston:Kluwer Academic Publishers, c1997． ,p.267-278
[67] Leifert,-C;Waites,-W.M. Bacterial growth in plant tissue culture media. J-Appl-Bacteriol. Oxford : Blackwell Scientific Publications. June 1992． v.72(6) p.460-466
[68] Leifer,-C.;Woodward,-S. Laboratory contamination management: the requirement for microbiological quality assurance. Plant-cell,-tissue-organ-cult. Dordrecht, The Nether lands: Leifert, C., Waites, W.M.&Nicholas, J.R. Bacterial contamination of micropropagated plant cultures. Journal of Applied Bacteriology, 1989a, 67:353-361
[69] Leifert, C., Waites, W.M., Camotta, H.& Nicholas, J.R. 1989 Lactobacillus plantarum: a deleterious contaminant of plant tissue culture. Journal of Applied Bacteriology 67, 363-370．
[70] Leifert, C., Waites, W. M., Camotta, H. & Nicholas, J.R. Lactobacillus plantarum ; a deleterious contamination of plant tissue culture. Journal of Applied Bacteriology, 1989b, 67:363-370
[71] Long, R.D., T.F. & Cassells, A.C An investigation of the effects of bacterial
contaminations on potato nodal cultures. Acta Horticulturae, 1988, 225:83-91
[72] Long,R.D.Curtin,T.R, and Cassells,A.C., 1988． An investigation into the effects of bacterial contaminants on potato nodal cultures. Acta Hort.225:83-91
[73] Leggat, I., Waites, W. M. Leifert. C. and Nictholas. J. R. 1988, Characterisation of micro-organisms isolated from plants during micropropagation. Acta Horticulutrae 225,93-102
[74] Lelliott, R. A. and Stead, D. E., 1987． Methods for the diagnosis of bacterial diseases of plants. Blackwell Scientific Publications Ltd. ISBN 0-632-01233-1．
[75] Leifert,-C.; Morris,-C.E.; Waites,-W.M. Ecology of microbial saprophytes and pathogens in tissue culture and field-grown plants:reasons for contamination problems in vitro. Crit-rev-plant-sci. Boca Raton, Fla.:CRC Press, [cl983-.1994． v.13(2) p.139-183．
[76] Mathias, P.J., Alderson, P.G. & Leakey, R.R.B. Bacterial contamination in tropical hardwood culturesss. Acta Horticulturae, 1987, 212: 43-49
[77] Mathias,R.J. and Boyd, L.A., 1986． Cefotaxime stimulates callus growth, embryogenesis and regeneration in hexaploid bread wheat(Triticum aestivum). Plant Science 46:217-223
[78] Mathias,P.J.,Alderson,P.G. and Leakey, R.R.B.,1985． Bacterial contamination in cultures of tropical hard wood species. Symposium: In vitro problems related to mass propagation of horticultural plants. Gembloux, 16-20 Steptember 1985．
[79] Macfaddin, J.F. 1980 Biochemical Tests for Identification of Medical Bacteria pp. 15-16． Baltimore: Williams and Wilkins.
[80] Niedz,-R. P. Using isothiazolone biocides to control microbial and fungal contaminants in plant tissue cultures. HortTechnology. Alexandria, VA: American Society for Horticultural Science, cl991-. Oct/Dec 1998． v.8 (4) p. 598-601
[81] Poulszn, G.B.(1988) Elimination of contamination microorganisms from meristem culture of apple rootstock M26． Acta Horticulturae,1989a, 225: 193-197
[82] Phillips, R., Aenott, S.N. & Kaptan, S.E. Antibiotics in plant tissue culture: Rifampicin effectively controls bacterial contaminants without affecting the growth of short-term explant cultures of Helianthus tuberosus. Plant Science Letters, 1981, 21:23 5-240
[83] Reustle, G. , Mann, .M. & Heintz, C. Experience and problems with infection in tissue culture of grapevine. Acta Horticulturae, 1988,225: 119-129
[84] Sharma-HC; Singh-SK; Goswami-AM; Singh-SP; Kishor-PBK. Plant tissue culture and biotechnology: emerging trends. Proceedings of a symposium held at Hyderabad, 29-31 Januaryl997, 1999: 151-155
[85] Szendrak,-E.;Read,-P.E.;Yang,-G.C. Prevention and elimination of contamination for in vitro culture of several woody species. Plant-cell,-tissue-organ-cult. Dordrecht, The Nether lands: Kluwer Academic Publishers,cl997． ,p.233-236
[86] Szendrak,-E.;Read,-P.E.;Yang,-G.C. Prevention and elimination of contamination for in vitro culture of several woody species. Plant-cell,-tissue-organ-cult. Dordrecht. The Nether lands: Kluwer Academic Publishers,cl997． ,p.233-236
[87] Schaad, N.W., 1980． Laboratory guide for identification of pathogenic bacteria. American Phytopathological Society, Minnesota.
[88] Schaad, N. W. 1980 Initial identification of common genera. In Identification of Plant Pathogenic Bacteria ed. Schaad, N. W. pp. 1-11． Paul: American Phytopathological Society.
[89] Schaad, N.W.,1980． Laboratory guide for identification of pathogenic bacteria. American Phytopathological Society , Minnesota.
[90] Trick, I. & Lingens, F. 1985 Aerobic spore-forming bacteria as detrimental infectants in plant tissue cultures. Applied Microbiology and Biotechnology 21, 245-249．
[91] Tanaka, M. , Kumura, M. and Goi.M. 1983, Surface sterilization for in vitro culture of Phalaenopsis Flower-stalk. Acta Horticulturae, 131, pp 321-328
[92] Williamson,-B;Cooke,-D.E.L., Fungal infections of micropropagated plants at weaning : a problem exemplified by downy mildews in Rubus and Rosa. Plant-cell,-tissue-organ-cult. Dordrecht, The Nether lands: Kluwer Academic Publishers. 1998． v.52(1/2) p.89-96
[93] Widholm,-J.M. The rare occurrence of tissue culture contamination by Methylobacterium mesophilicum. Plant-cell,-tissue-organ-cult. Dordrecht, The Nether lands: Kluwer Academic Publishers. June 1996． v.45(3) p.201-205
[94] Wilson,Z.A. and Power, J.B. 1989． Elimination of systemic contamination in explant and protoplast cultures of rubber (Hevea brasiliensis Muell.Arg.).Plant Cell Rep.7:622-625
[95] Wilson,Z.A. and Power, J.B.1989． Elimination of systemic contamination in explant and protoplast cultures of rubber (Hevea brasiliensis Muell.Arg.).Plant Cell Rep.7:622-625
[96] Young,P.M., Hutchins, A.S. and Canfield, M.L.,1984． Use of antibiotics to control bacteria in shoot cultures of woody plants. Plant Science Letters 34: 203-209
[97] Young, P.M., Hutchins, A.S.& Canfield, M.L. Use of antibiotics to control bacteria in shoot cultures of woody plants. Plant Science Letters, 1984, 34: 203-209
[98] Young, P.M., Hutchins, A.S. and Canfield, M.L., 1984． Use of antibiotics to control bacteria in shoot cultures of woody plants. Plant Science Letters 34:203-209．
[2] 朱广廉．植物组织培养中灭菌和无菌操作的几个问题，植物生理学通讯，1995，31(5)：373-375
[3] 朱广廉．植物组织培养中的外植体灭菌．植物生理学通讯，1996，32(6)：444-449
[4] 林盛，马崇烈．组织培养中污染的控制．广西农业科学，1994(2)：87-88
[5] 何云芳，余有祥．金线菊与菊芋在组织培养中细菌污染的防治，浙江林业科技，1997(4)：18-22
[6] 王玉军．山东农业大学硕士研究生毕业论文，2000。
[7] 杨红花．山东农业大学硕士研究生毕业论文，2001。
[8] 王心钗编．植物显微技术[M]，1986，福建教育出版社。
[9] 邹文欣，谭仁祥．植物内生菌研究新进展，植物学报，2001，43(9)：881-892
[10] 韩美丽，陆荣生等．绿巨人组培继代过程中玻璃化苗及细菌污染的消除方法研究，广西林业科学，1999，28(1)：16-19
[11] 蔡静，王亚静等．O_3消毒机与紫外线消毒比较，湖北医科大学学报，1999，20(3)：263-264
[12] 韩玉坤，周华等．臭氧与紫外线对医院病房空气消毒效果比较，中国消毒学杂志，1997，14(3)：185
[13] 奏启玲，贾瑞萍等．医院内三种空气消毒方法比较，齐鲁护理杂志，1999，5(3)
[14] 波边一功，陈微微．抗真菌抗生素，国外医药：抗生素分册，1989，10(5)：377-379
[15] 翟建中，顾梅俏．长春蔓组培生产中污染菌的防除，森林病虫通讯，1999，4：30-32
[16] 肖显华，王顺珍等．植物材料表面消毒方法的改进，生物技术，1999，9(1)：43-45
[17] 黄惜等．抗生素在防止香蕉组培苗污染上的应用研究，海南岛农业科技，1995，(2)：18-20
[18] 谭文澄，戴策刚．观赏植物组织培养技术，1997。
[19] 井上真由美．微生物灾害及其防止技术，上海科学技术出版社。
[20] 邵力平，沈瑞祥等．真菌分类学，中国林业出版社。
[21] 魏景超．真菌鉴定手册，上海科学技术出版社。
[22] 山东农业大学，莱阳农学院编，微生物学试验，北京农业大学出版社。
[23] 曹孜义等．编实用植物组培培养技术教程，1996，甘肃科学技术出版社。
[24] 沈惠娟．木本植物组织培养技术，1992，中国农业科学出版社。
[25] 张开旺．抗生素对大豆组织培养物的抑制效应，1991(1)：45-47
[26] 杨颐康．微生物学，1987，高等教育出版社。
[27] 陈正华主编．木本植物组织培养及其应用，1986，高等教育出版社。
[28] 许婉芳，龚福生等．杀菌剂对金线莲组织培养中微生物污染的抑制作用，福建果树，1999(110)：6-7
[29] 张贯新，晋萍．臭氧用于空气消毒效果观察，中原医刊，1999，26(6)：32-33
[30]陈华癸、樊庆笙主编的《微生物学》，1991
[31]任丽霞，秦香丽，浅谈手术室交叉感染的预防和控制，山西临床医药杂志，1999 6(3)：211-212
[32]Boxus, P.H.& Terzi, J.-M. Control of accidental contamination during mass propagation. Acta Horticulturae, 1988,225:189-193
[33]Blake, J. Mites and thrips as bacterial and fungi vectors between plant tissue cultures. Acta Hortuculturae ,1988, 225:163-166
[34]Boxus,Ph. And Terzi, J.M.,1987. Big losses due to bacterial contaminations can be avoided in mass propagation scheme. Acta Hortic.212(1): 91-93
[35]Bhansali, R.R. and Singh, k., 1986. In vitro emergence of multiple plantlets from smut infected apical tips of sugareane nodal buds. Indian Phytopathol, 39:582-583.
[36]Brenner, D.J.,1984 Description of the species in the family Enterobacteriaceae. In Bergey's Manual of Systematic Bacteriology ed. Krieg,N.R.&Holt, J.G.pp.408-420. Baltimore:Williams and Wilkins.
[37]Bastiaens, L., 1983. Endogenous bacteria in plants and their implications in tissue culture. A review Med. Fac. Landbourw. Rijksuniv. Gent. 48/1:1-11.
[38]Constantine, D. R., 1986. Micropropagation in the commercial environment. In: Plant Tissue Culture and Agricultural Applications (eds. L. A. Withers and P.G. Alderson). Butterworth, London. Journal-of-Agriculture-and-Forestry. 1999, 48: 4, 79-91
[39]Chen-ChiaChung; Huang-ChingWen; Chen-CC; Huang-CW Cotty,-P.J. Method for the control or prevention of aflatoxin contamination using a non-toxigenic strain of Aspergillus niger flavus. United states Department of Agriculture patents. [Washington, D.C.?:The Department, 1900?-.Mar15,1994.(5,294,442) 1 p.88-92
[40]C. Lelfert. et al. Eilmation of Lactobacillus plantarum,Corynebacterium spp.,Staphylococcus saprophyticus and Pseudomonas paucimobills from micropropagated Hemerocallis,Cholsya and Delphinium cultures using antibiotics. Jurnal of Applied Bacteriology, 1991,71:307—330
[41]Casseus.A.c problem in tissue culture : culture contamination in micropropagation technology and application[M]. Kluwer. Academic publishers. 1991, 31-34
[42]Cornu, D.& Michel, M.F. Bacteria contamination in shoot cultures of Prunus avium L. choice and phytotoxicity of antibiotics. Acta
Horticulurae ,1987,212:83-86
[43] Constantine, D. R. , 1986． Micropropagation in the commercial environment. In: Plant Tissue Culture and its Agricultural Applications (eds., L. A. Withers and P. G. Alderson). ISBN 0-407-00921-3．
[44] Cassells, A.C. and Long, R.D., 1982． The elimination of potato viruses X,Y,S and M in meristem and explant cultures of potato in the presence of Virazole. Potato Res. 25: 165-173
[45] de Fossard, R.A. and Watanabe.I., 1988． Development of a photoautotrophic tissue culture system for shoots and /or plantlets at rooting and acclimatization stages. Acta Horticulturae 230:153-158
[46] Dahiya, J.S., Woods, D.L., and Tewari, J. P., 1988． Control of Rhizoctonia solani, causal agent of brown girding root rot of rapeseed, by Pseudomonas fluorescens. Bot. Bull. Acad. Sinica 29: 135-142．
[47] Davison, J., 1988． Plant beneficial bacteria. Biotechnology 6:282-286．
[48] Debergh, P.C.& Vandershaeghe, A.M. Some symptoms indicating the presence of bacterial contaminations in plant tissue cultures. Acta Horticulturae, 1988, 225:77-83
[49] Debergh, P. and Maene,L., 1984． Pathological and physiological problems related to the in vitro culture of plants. Parasitica 40/2-3:69-75．
[50] Dychdala, G.R., 1983． Chlorine and chlorine compounds. In: Disinfection, Sterilization and Preservation. 3rd Edition (ed. S.S. Block). Lea and Febiger, Philadelphia: 157-182．
[51] Edvvin B. Herman, Non-axenic plant tissue culture: possibilities and opportunities. Acta horticulturae, 1990, 280:1990．
[52] Enjalric,F.,Carron, M.P.& Lardet, L. Contamination of primary cultures in tropical areas: The case of Hevea brasiliensis. Acta Horticulturae ,1988,225:57-65
[53] Enjalric,F..1986． Le probleme des infections en zone subtropicale humide. Rapport IRCA/ClRAD3/86:14p.
[54] Falkiner,-F.R. Antibiotics in plant tissue culture and micropropagation-what are we aiming at. Pathogen and microbial contamination management in micropropagation/Dordrecht; Boston:Kluwer Academic Publishers, c1997． ,155-160
[55] Falkiner, F.R Strategy for the selection of antibiotics for use against common bacterial pathogens and endophytes of plants. Acta Horticulturae., 1988,225:53-57
[56] Fisse,J., Batalle, A. & Pera, J. Endogenous bacteria elimination in ornamental plants. Acta Horticulturae, 1987, 212: 87-90
[57] Fisse, J.. Batlle, A. and Pera, J., 1985． Endogenous bacteria elimination in ornamental explants. Symposium: In vitro problems related to mass propagation horticultural plants. Gembloux 16-20 Stember 1985．
[58] Holford.-P.;Newbury,-H.J. The effects of antibiotics and their breakdown products on
the in vitro growth of Antirrhinum majus. Plant-Cell-Rep. Berlin, W. Ger.: Springer International. 1992． V.11(2) p.93-96
[59] Herman, E.B., Toward control of micropagation contamination. Agericell Report, 19879:33-35
[60] Herman, E.B., 1987． Toward control of micropropagation contamination. Agricell Report 9: 33-35．
[61] Juhnke, M.E., Mathre, D.E., and Sands, D.C., 1987． Identification and characterization of rhizosphere-competent bacteria of wheat. Appl. Environ. Microbiol.53:2793-2799．
[62] Jong,S.C., 1987． Prevention and control of mite infestations in fungus cultures. American Type Culture Collection (ATCC) Quarterly Newsletter 7(1) : 1 and 7．
[63] Kelman, A. & Dickey, R.S. 1980 Soft rot or 'carotophora'group. In Identification of Plant Pathogenic Bacteria ed. Schaad, N.W. pp.31-35． Paul: American Phytopathological Society.
[64] Langens-Gerrits,-M; Albers,-M.; Klerk,-G.J.D. Hot-water treatment before tissue culture reduces initial contamination in Lilium and Acer. Plant-cell,-tissue-organ-cult. Dordrecht, The Nether lands: Kluwer Academic Publishers. 1998． v.52( 1/2) p. 83-88
[65] Leggatt, I., Waites, W. M. Leifert, C. & Nicholas, J. R. (1988) Characterisation of micro-organisms isolated from plants during micropropagation. Acta Horticulturae, 1989a, 225: 93-102 Kluwer Academic Publishers. 1998． v.52(1/2) p.83-88
[66] Long,-R.D. Photoautotrophic micropropagation-a strategy for contamination control. Pathogen and microbial contamination management in micropropagation/Dordrecht;Boston:Kluwer Academic Publishers, c1997． ,p.267-278
[67] Leifert,-C;Waites,-W.M. Bacterial growth in plant tissue culture media. J-Appl-Bacteriol. Oxford : Blackwell Scientific Publications. June 1992． v.72(6) p.460-466
[68] Leifer,-C.;Woodward,-S. Laboratory contamination management: the requirement for microbiological quality assurance. Plant-cell,-tissue-organ-cult. Dordrecht, The Nether lands: Leifert, C., Waites, W.M.&Nicholas, J.R. Bacterial contamination of micropropagated plant cultures. Journal of Applied Bacteriology, 1989a, 67:353-361
[69] Leifert, C., Waites, W.M., Camotta, H.& Nicholas, J.R. 1989 Lactobacillus plantarum: a deleterious contaminant of plant tissue culture. Journal of Applied Bacteriology 67, 363-370．
[70] Leifert, C., Waites, W. M., Camotta, H. & Nicholas, J.R. Lactobacillus plantarum ; a deleterious contamination of plant tissue culture. Journal of Applied Bacteriology, 1989b, 67:363-370
[71] Long, R.D., T.F. & Cassells, A.C An investigation of the effects of bacterial
contaminations on potato nodal cultures. Acta Horticulturae, 1988, 225:83-91
[72] Long,R.D.Curtin,T.R, and Cassells,A.C., 1988． An investigation into the effects of bacterial contaminants on potato nodal cultures. Acta Hort.225:83-91
[73] Leggat, I., Waites, W. M. Leifert. C. and Nictholas. J. R. 1988, Characterisation of micro-organisms isolated from plants during micropropagation. Acta Horticulutrae 225,93-102
[74] Lelliott, R. A. and Stead, D. E., 1987． Methods for the diagnosis of bacterial diseases of plants. Blackwell Scientific Publications Ltd. ISBN 0-632-01233-1．
[75] Leifert,-C.; Morris,-C.E.; Waites,-W.M. Ecology of microbial saprophytes and pathogens in tissue culture and field-grown plants:reasons for contamination problems in vitro. Crit-rev-plant-sci. Boca Raton, Fla.:CRC Press, [cl983-.1994． v.13(2) p.139-183．
[76] Mathias, P.J., Alderson, P.G. & Leakey, R.R.B. Bacterial contamination in tropical hardwood culturesss. Acta Horticulturae, 1987, 212: 43-49
[77] Mathias,R.J. and Boyd, L.A., 1986． Cefotaxime stimulates callus growth, embryogenesis and regeneration in hexaploid bread wheat(Triticum aestivum). Plant Science 46:217-223
[78] Mathias,P.J.,Alderson,P.G. and Leakey, R.R.B.,1985． Bacterial contamination in cultures of tropical hard wood species. Symposium: In vitro problems related to mass propagation of horticultural plants. Gembloux, 16-20 Steptember 1985．
[79] Macfaddin, J.F. 1980 Biochemical Tests for Identification of Medical Bacteria pp. 15-16． Baltimore: Williams and Wilkins.
[80] Niedz,-R. P. Using isothiazolone biocides to control microbial and fungal contaminants in plant tissue cultures. HortTechnology. Alexandria, VA: American Society for Horticultural Science, cl991-. Oct/Dec 1998． v.8 (4) p. 598-601
[81] Poulszn, G.B.(1988) Elimination of contamination microorganisms from meristem culture of apple rootstock M26． Acta Horticulturae,1989a, 225: 193-197
[82] Phillips, R., Aenott, S.N. & Kaptan, S.E. Antibiotics in plant tissue culture: Rifampicin effectively controls bacterial contaminants without affecting the growth of short-term explant cultures of Helianthus tuberosus. Plant Science Letters, 1981, 21:23 5-240
[83] Reustle, G. , Mann, .M. & Heintz, C. Experience and problems with infection in tissue culture of grapevine. Acta Horticulturae, 1988,225: 119-129
[84] Sharma-HC; Singh-SK; Goswami-AM; Singh-SP; Kishor-PBK. Plant tissue culture and biotechnology: emerging trends. Proceedings of a symposium held at Hyderabad, 29-31 Januaryl997, 1999: 151-155
[85] Szendrak,-E.;Read,-P.E.;Yang,-G.C. Prevention and elimination of contamination for in vitro culture of several woody species. Plant-cell,-tissue-organ-cult. Dordrecht, The Nether lands: Kluwer Academic Publishers,cl997． ,p.233-236
[86] Szendrak,-E.;Read,-P.E.;Yang,-G.C. Prevention and elimination of contamination for in vitro culture of several woody species. Plant-cell,-tissue-organ-cult. Dordrecht. The Nether lands: Kluwer Academic Publishers,cl997． ,p.233-236
[87] Schaad, N.W., 1980． Laboratory guide for identification of pathogenic bacteria. American Phytopathological Society, Minnesota.
[88] Schaad, N. W. 1980 Initial identification of common genera. In Identification of Plant Pathogenic Bacteria ed. Schaad, N. W. pp. 1-11． Paul: American Phytopathological Society.
[89] Schaad, N.W.,1980． Laboratory guide for identification of pathogenic bacteria. American Phytopathological Society , Minnesota.
[90] Trick, I. & Lingens, F. 1985 Aerobic spore-forming bacteria as detrimental infectants in plant tissue cultures. Applied Microbiology and Biotechnology 21, 245-249．
[91] Tanaka, M. , Kumura, M. and Goi.M. 1983, Surface sterilization for in vitro culture of Phalaenopsis Flower-stalk. Acta Horticulturae, 131, pp 321-328
[92] Williamson,-B;Cooke,-D.E.L., Fungal infections of micropropagated plants at weaning : a problem exemplified by downy mildews in Rubus and Rosa. Plant-cell,-tissue-organ-cult. Dordrecht, The Nether lands: Kluwer Academic Publishers. 1998． v.52(1/2) p.89-96
[93] Widholm,-J.M. The rare occurrence of tissue culture contamination by Methylobacterium mesophilicum. Plant-cell,-tissue-organ-cult. Dordrecht, The Nether lands: Kluwer Academic Publishers. June 1996． v.45(3) p.201-205
[94] Wilson,Z.A. and Power, J.B. 1989． Elimination of systemic contamination in explant and protoplast cultures of rubber (Hevea brasiliensis Muell.Arg.).Plant Cell Rep.7:622-625
[95] Wilson,Z.A. and Power, J.B.1989． Elimination of systemic contamination in explant and protoplast cultures of rubber (Hevea brasiliensis Muell.Arg.).Plant Cell Rep.7:622-625
[96] Young,P.M., Hutchins, A.S. and Canfield, M.L.,1984． Use of antibiotics to control bacteria in shoot cultures of woody plants. Plant Science Letters 34: 203-209
[97] Young, P.M., Hutchins, A.S.& Canfield, M.L. Use of antibiotics to control bacteria in shoot cultures of woody plants. Plant Science Letters, 1984, 34: 203-209
[98] Young, P.M., Hutchins, A.S. and Canfield, M.L., 1984． Use of antibiotics to control bacteria in shoot cultures of woody plants. Plant Science Letters 34:203-209．