分蘖洋葱病毒病原鉴定及脱毒苗培养·增殖技术的研究
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摘要
黑龙江省分蘖洋葱病毒病发生普遍，阿城、宾县、五常三个地区的发病率均为100％，
病情指数分别为62.13％、71.07％和49.87％。典型症状为黄化条纹花叶，叶片扭曲畸形、
叶尖干枯，植株矮缩，鳞茎退化变小，产量和品质大幅度下降。
人工接种发现，可被分蘖洋葱病毒系统感染的植物有洋葱、大葱、大蒜和蚕豆；局
部感染的有千日红、苋色藜。电镜观察表明，病体细胞中含有大量线状病毒粒体，其长
度范围在300～1450nm之间。通过叶片超薄切片，在叶肉细胞质中观察到大量的风轮状、
束状、涡轮状和环状内含体，叶绿体被破坏。体外抗性测定结果：致死温度为55～65℃，
稀释限点为10~(-4)，体外保毒期为3d。酶联免疫测试和洋葱黄矮病毒抗血清、大蒜复合病
毒抗血清呈阳性反应。根据以上特征证明，黑龙江省分蘖洋葱病毒病为几种病毒复合侵
染，具体包括哪几种有待于进一步研究。
以阿城紫皮分蘖洋葱鳞茎0.15～0.3mm茎尖为外植体，以MS为基本培养基，添加蔗
糖3.0％，琼脂0.8％，pH值调至5.7～5.8。通过激素配比试验，筛选出最佳的培养基组成。
利用植物组织培养技术，获得了分蘖洋葱无毒试管苗，结果表明：最佳诱芽培养基为
MS+BA0.1mg/l+NAA0.4mg/l，诱导率为100％，平均每个茎尖外植体形成3.1个不定芽，
而且无愈伤组织的形成；最佳生根培养基为1/2MS+IBA1.5mg/l+NAA0.01mg/l+PP_(333)0.1
mg/l，利用此项组培技术，进行了不同外植体大小、鳞茎热处理对脱毒效果影响的研究，
结果表明，80℃高温处理10min，然后剥取0.3～0.5mm的茎尖进行培养，脱毒率为100％。
以阿城紫皮分蘖洋葱鳞茎茎尖为外植体材料，对分蘖洋葱愈伤组织的诱导、分化、
再生苗的生根和移栽进行了研究。结果表明：（1）在不同激素组合的培养基上分蘖洋葱的
茎尖均能产生愈伤组织，其中2,4-D2.0mg/l+KT0.5mg/L的MS培养基对愈伤组织的诱导
最有利，其出愈率为100％；（2）在愈伤组织的分化中，转接到添加NAA、BA的MS分
化培养基上，愈伤组织进一步膨大，出现绿色芽点，均形成大量的丛生芽，其中以NAA0.1
mg/l+BA0.4mg/l的MS分化培养基最佳，其分化率为88.33％。平均成苗数为10.2，技
术上达到了快速繁殖、规模生产的要求；（3）再生植株生根和壮苗的最佳激素组合为
1/2MS+PP_(333)0.1mg/l+NAA0.01mg/l+IBA1.5mg/l，生根率为100％，根系发达，叶色浓绿，
生长健壮。移栽后长势良好，对环境具有较强的适应性，移栽成活率达100％。
分蘖洋葱试管微鳞茎的诱导研究表明：离体条件下，温度和光周期对试管鳞茎的形
成起着决定性的作用，而且提高培养基的pH值、增加蔗糖浓度也有重要的调控作用。
细胞学观察表明：茎尖分生组织培养脱毒苗遗传稳定，染色体未发生变异，均为
2n=16；愈伤组织及其再生苗遗传稳定性差，愈伤组织染色体变异率为33.85％，其中单
倍体占9.23％，四倍体占15.39％，非整倍体占9.23％；愈伤组织再生苗染色体变异率为
24.62％，其中四倍体占16.93％，非整倍体占7.69％。未发现三倍体的存在。
通过以上研究，对分蘖洋葱建立了有效的茎尖培养脱毒苗的速繁体系。
Almost all Tillered-onion ( A/hum. cepa L. var. mu/tip/cans Bailey syn. Var. Agrogatum Don)being cultured in Heilongjiang province are infected by viruses. Hundred percent of Tillered-onion plants in Acheng, Binxian and Wuchang county of 1-leilongjiang were infected by viruses, the index is 62.13%, 71.07% and 49.87% respectively. The infection induces typical mosaic symptoms of yellow streak as well as curling on leaves, withering of leaf-tips, dwarfism on the plants, and resulting in the degeneration and reduction of size of bulbs. Virus infection produces important reductions of quality and yield in Tillered-onion.
This disease was demonstrated to be caused by several viruses which could be transmitted by the sap of the diseased plants. A.cepa L , A Ilium sativum, Ahhiumfistulosum L. and vicia faba were infected systemically by sap inoculation, Local lesions were produced on the inoculated leaves of Che. A,naranticola and Gomphrena globosa. Electron microscopic observations of the natually infected Tillered-onion revealea that there are fiexuous filamentous particles 300?450nm long, with 500?00nm as the norm. 32.67% are 550~?50nm, 9%are 650?00nm and 3扵.6~T%are 700~800nm in particles, About 80% of the particles in the range of 550?00nrn. Typical pinwheel type, bundle, scrool and circular cytoplasmic inclusions are found in the cytoplasm of the mesophyll cell could be observed by ultrathin section of leaves, the chlorophylls are destroied. In sap ,it properties were thermal inactivation point (TIP) between55~?5~C,dilution end point (DEP) was l0-~, long in vitro (LIV) of extract at room temperature was 3d. Indirec
t-ELISE of leaf extracts of Tillered-onion plants showed positive reaction with the antisera against onion yellow dwarf virus, complex garlic viruses. Evidence from study, Heilongjiang Tillered-onion is jointly infected by several kinds of viruses, but need to be identified continuelly.
Virus-free plants of tillered-onion were obtained from in vitro meristem tip apex tissue culture with the basical medium of MS. Each of medium was added 3.0%sucrose and 0.8% agar and the medium pH value is 5.7~5.8. Aseries of optimization experiment for cultural medium composition, concentration of phytohormones were investgated with a view to acclerate the propagation of virus0free plants. The result of virus elimination by meristem tip culture of purpie-skined Tillered-onion commonly grown in Acheng showed that the best medium shooting wasMS+NAAO.lmg/l+BAO.4mg/l, On average, 3.lshoots formed from one meristem tip without callus formation. and that for rooting was 1/2 MS+NAAO.Olmg/I+IBAI.5mg/l+PP3330.l mg/I. Effects of virus-free culture with different meristem tips in size and heated-treated are discussed. The result showed that
pretreatment of cloces with 80~Cthermotheray for 10mm, the obtained virus-free plants by using 0.3~?.5mm meristem tip.
This paper deals with studies on in vitro culture of meristem tip of Tillered-onion, including callus induction and differentiation, plantlet root, development and transplantation. Results showed: (1) callus were obtained on media containing different kinds of phytohormone.lt was advantageous to callus induction on MS basal medium supplemented with 2,4-D2.Omg/l and KTO.Smg/l. the frequency of callus induction was 100%; (2) The combination of NAAO.lmg/l and BAO.4mg/l was best medium for callus differentiation of which ratio was 88.33%, Most of callus was aggrandized again and induced green bud spots then formed multiplying clumpy buds, the mean number of plantlets per callus was 10.2, These results could meet the technology requirements of rapid propagation in large scale production; (3) 100% of the regenerated plantlets treated with NAAO.Olmg/l and IBA1.Smg/l and PP3330.1 mg/I could develop their roots, grew robustly and robust green leaves, after the regenerated plantlets were transplanted to soil, they co
uld adapt to environment. The survival ratio of transplanting reached 100%.
Incubation temperture and photoperiod were essential re
病情指数分别为62.13％、71.07％和49.87％。典型症状为黄化条纹花叶，叶片扭曲畸形、
叶尖干枯，植株矮缩，鳞茎退化变小，产量和品质大幅度下降。
人工接种发现，可被分蘖洋葱病毒系统感染的植物有洋葱、大葱、大蒜和蚕豆；局
部感染的有千日红、苋色藜。电镜观察表明，病体细胞中含有大量线状病毒粒体，其长
度范围在300～1450nm之间。通过叶片超薄切片，在叶肉细胞质中观察到大量的风轮状、
束状、涡轮状和环状内含体，叶绿体被破坏。体外抗性测定结果：致死温度为55～65℃，
稀释限点为10~(-4)，体外保毒期为3d。酶联免疫测试和洋葱黄矮病毒抗血清、大蒜复合病
毒抗血清呈阳性反应。根据以上特征证明，黑龙江省分蘖洋葱病毒病为几种病毒复合侵
染，具体包括哪几种有待于进一步研究。
以阿城紫皮分蘖洋葱鳞茎0.15～0.3mm茎尖为外植体，以MS为基本培养基，添加蔗
糖3.0％，琼脂0.8％，pH值调至5.7～5.8。通过激素配比试验，筛选出最佳的培养基组成。
利用植物组织培养技术，获得了分蘖洋葱无毒试管苗，结果表明：最佳诱芽培养基为
MS+BA0.1mg/l+NAA0.4mg/l，诱导率为100％，平均每个茎尖外植体形成3.1个不定芽，
而且无愈伤组织的形成；最佳生根培养基为1/2MS+IBA1.5mg/l+NAA0.01mg/l+PP_(333)0.1
mg/l，利用此项组培技术，进行了不同外植体大小、鳞茎热处理对脱毒效果影响的研究，
结果表明，80℃高温处理10min，然后剥取0.3～0.5mm的茎尖进行培养，脱毒率为100％。
以阿城紫皮分蘖洋葱鳞茎茎尖为外植体材料，对分蘖洋葱愈伤组织的诱导、分化、
再生苗的生根和移栽进行了研究。结果表明：（1）在不同激素组合的培养基上分蘖洋葱的
茎尖均能产生愈伤组织，其中2,4-D2.0mg/l+KT0.5mg/L的MS培养基对愈伤组织的诱导
最有利，其出愈率为100％；（2）在愈伤组织的分化中，转接到添加NAA、BA的MS分
化培养基上，愈伤组织进一步膨大，出现绿色芽点，均形成大量的丛生芽，其中以NAA0.1
mg/l+BA0.4mg/l的MS分化培养基最佳，其分化率为88.33％。平均成苗数为10.2，技
术上达到了快速繁殖、规模生产的要求；（3）再生植株生根和壮苗的最佳激素组合为
1/2MS+PP_(333)0.1mg/l+NAA0.01mg/l+IBA1.5mg/l，生根率为100％，根系发达，叶色浓绿，
生长健壮。移栽后长势良好，对环境具有较强的适应性，移栽成活率达100％。
分蘖洋葱试管微鳞茎的诱导研究表明：离体条件下，温度和光周期对试管鳞茎的形
成起着决定性的作用，而且提高培养基的pH值、增加蔗糖浓度也有重要的调控作用。
细胞学观察表明：茎尖分生组织培养脱毒苗遗传稳定，染色体未发生变异，均为
2n=16；愈伤组织及其再生苗遗传稳定性差，愈伤组织染色体变异率为33.85％，其中单
倍体占9.23％，四倍体占15.39％，非整倍体占9.23％；愈伤组织再生苗染色体变异率为
24.62％，其中四倍体占16.93％，非整倍体占7.69％。未发现三倍体的存在。
通过以上研究，对分蘖洋葱建立了有效的茎尖培养脱毒苗的速繁体系。
Almost all Tillered-onion ( A/hum. cepa L. var. mu/tip/cans Bailey syn. Var. Agrogatum Don)being cultured in Heilongjiang province are infected by viruses. Hundred percent of Tillered-onion plants in Acheng, Binxian and Wuchang county of 1-leilongjiang were infected by viruses, the index is 62.13%, 71.07% and 49.87% respectively. The infection induces typical mosaic symptoms of yellow streak as well as curling on leaves, withering of leaf-tips, dwarfism on the plants, and resulting in the degeneration and reduction of size of bulbs. Virus infection produces important reductions of quality and yield in Tillered-onion.
This disease was demonstrated to be caused by several viruses which could be transmitted by the sap of the diseased plants. A.cepa L , A Ilium sativum, Ahhiumfistulosum L. and vicia faba were infected systemically by sap inoculation, Local lesions were produced on the inoculated leaves of Che. A,naranticola and Gomphrena globosa. Electron microscopic observations of the natually infected Tillered-onion revealea that there are fiexuous filamentous particles 300?450nm long, with 500?00nm as the norm. 32.67% are 550~?50nm, 9%are 650?00nm and 3扵.6~T%are 700~800nm in particles, About 80% of the particles in the range of 550?00nrn. Typical pinwheel type, bundle, scrool and circular cytoplasmic inclusions are found in the cytoplasm of the mesophyll cell could be observed by ultrathin section of leaves, the chlorophylls are destroied. In sap ,it properties were thermal inactivation point (TIP) between55~?5~C,dilution end point (DEP) was l0-~, long in vitro (LIV) of extract at room temperature was 3d. Indirec
t-ELISE of leaf extracts of Tillered-onion plants showed positive reaction with the antisera against onion yellow dwarf virus, complex garlic viruses. Evidence from study, Heilongjiang Tillered-onion is jointly infected by several kinds of viruses, but need to be identified continuelly.
Virus-free plants of tillered-onion were obtained from in vitro meristem tip apex tissue culture with the basical medium of MS. Each of medium was added 3.0%sucrose and 0.8% agar and the medium pH value is 5.7~5.8. Aseries of optimization experiment for cultural medium composition, concentration of phytohormones were investgated with a view to acclerate the propagation of virus0free plants. The result of virus elimination by meristem tip culture of purpie-skined Tillered-onion commonly grown in Acheng showed that the best medium shooting wasMS+NAAO.lmg/l+BAO.4mg/l, On average, 3.lshoots formed from one meristem tip without callus formation. and that for rooting was 1/2 MS+NAAO.Olmg/I+IBAI.5mg/l+PP3330.l mg/I. Effects of virus-free culture with different meristem tips in size and heated-treated are discussed. The result showed that
pretreatment of cloces with 80~Cthermotheray for 10mm, the obtained virus-free plants by using 0.3~?.5mm meristem tip.
This paper deals with studies on in vitro culture of meristem tip of Tillered-onion, including callus induction and differentiation, plantlet root, development and transplantation. Results showed: (1) callus were obtained on media containing different kinds of phytohormone.lt was advantageous to callus induction on MS basal medium supplemented with 2,4-D2.Omg/l and KTO.Smg/l. the frequency of callus induction was 100%; (2) The combination of NAAO.lmg/l and BAO.4mg/l was best medium for callus differentiation of which ratio was 88.33%, Most of callus was aggrandized again and induced green bud spots then formed multiplying clumpy buds, the mean number of plantlets per callus was 10.2, These results could meet the technology requirements of rapid propagation in large scale production; (3) 100% of the regenerated plantlets treated with NAAO.Olmg/l and IBA1.Smg/l and PP3330.1 mg/I could develop their roots, grew robustly and robust green leaves, after the regenerated plantlets were transplanted to soil, they co
uld adapt to environment. The survival ratio of transplanting reached 100%.
Incubation temperture and photoperiod were essential re
引文
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[42] 扬春啸．克东毛葱销往越南．黑龙江日报，2000年1月4日第一版
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[44] 张西林等．见：中国植物生理学会，中国细胞生物学会编．全国植物组织和细胞培养及其应用 专题学术会议论文摘要汇编，1992，23
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[46] 赵成章等．多效唑对水稻未成熟胚愈伤组织诱导、分化和壮苗培养的影响．生物学报，1990， 32(5) ：407-409
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[51] 中科院上海植物生理研究所．植物组织与细胞培养.上海：上海科学技术出版社，1978：135-138．
[52] [美]G．N．Agrio著，陈永萱，陆家云，许志刚译．植物病理学(第三版)．北京：中国农业出版社， 1999：336-381
[53] 大岛信行．Recent studies of Antiphytoviral Substance．植物防疫，1993，47(3) ：25-29
[54] Ananthakrishman，T．N．．Thrips，chap，qinvectors of plant pathogens(Harris，K．F．and Maramorosch，K．Eds)Academic Press，NewYork，1980
[55] Baker，R．，Phytopathology，1962，52：1244-1255
[56] Baker，R．，etd．In：Kruse，P．E．J，Pallerson，M．K．J，et al．Tissue Culture：Methods and Application． NewYork：AcademicPress，1973：735
[57] Baker，R．．phytopathology，1962，52：1242-1244
[58] B．D．Harrison．生物技术方法在植物病毒流行学中的作用．国外农学-植物保护，1988(1) ：12-14
[59] Bhojwani，S．S．，Razdan，N．K．．Plant Tissue Culture Theory and Practice．Amsterdam，Elsevier， 1983，287
[60] Blaek，L．M．and Brakke，M．．Phytopathol．1952；42：269-273
[61] Bos，L．，Huijberts，N．，and Maat，Z．．Leek yellow stripe virus and relationships to onion yellow
virus, characterization, eology and possible control. Neth.J.Pl. path, 1978, 84: 185-204
[62] Bos,L.. Onion yellow dwarf virus. CMI/AAB Descriptions of plant viruses, I 976, No158,49
[63] Bos,L.. Viruses and virus disease of Allium species. Acta Horticulture, 1982,127:11-29
[64] Cassells, A. C. , Long,R.D. . Potato Res, I 982,251 : 65-173
[65] Choi,J.N., S ong,J.T.. Song, S. I., et al. Immunologic detection of garlic latent virus. Agricultural chemistry and Biotechnology, 1995,38( 1 ):49-54
[66] Derrick, K. S.. Virolog. 1973, 56:652-653
[67] Fan,Y., Wu,S., Lu,Z., et al. Molecular cloning and nucleotide sequence of the coat protein gene from garlic mosaic virus. Virologica Sinica, 1994,9(4) :333-340
[68] Fraenkel-Conrat,H.. The viruses catalogue, characterization and classification. 1985, P1 07
[69] Goheen,A.C., etal. Heat therapy of virus disease of perennial plants. Annupov,phytopat Hal, 1969 (7) : 331-345
[70] Hohne, C, . Schuster,G. , Frommhold,I. . Growth Regulatory Effects of 5-Azadihydrouracil and Ribavirin . Biochem Physiol Pflanzen, 1987,182:375-384
[71] Hollings,M., Stone, O. M.. Ann. Appl Biol, 1965,56: 73-86
[72] Kasugai,H., et al. Virucide Composition. Japan Kokai 78,75,325(CLA01N9/08) 04 July, 1978 Appl. 76/151,406, 16Doc, 1976, p6
[73] Kiho,Y. , Ahe,T. , Ohashi,Y. . Disassembly of Tobacco Mosaic Virus by Membrance Lipid Isolated from Tobacco Leaves and Polyornithine. Microbiol Immunol, 1979,23:735-748
[74] Lee, Y.W. , Yamazaki, T. , And lnouye,T. . Two elogated viruses in garlic, garlic latent virus and garlic mosaic virus. Nann. Phytoph. Soc. Japan, 1979,45: 727-734 .
[75] MachDonald, D. M.. Potato Res. 1973,16:263-269
[76] Matthews, R. E.F., plant virology(third edition). Academic Press, NewYork, 1991
[77] Mullin, R.H., et al. Phytopathology, 1974,64:1425-1429
[78] Mohamed,N.A. and Young, B.R.. Garlic yellow streak virus. a potyvirus infecting garlic in New Zealand. Ann. Appl. Biol. 1981,97: 65-74
[79] Pena-Iglesian,A.. Characterization of Spanish garlic viruses and their elimination by in vitro shoot apex culture. Acta Horticulture, 1982, 127: 183-193
[80] Zimmerman ,R. H., 朱玉林译．意大利的草莓苗木工厂．国外农学-果树, 1981 (4) : 24
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