晚香玉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
黑龙江省栽培的晚香玉单瓣品种的病毒发病率较低，一般为45.95％左右，重瓣品种中Poarl的发病率最高，达到97.3％。发病植株普遍出现叶面嵌纹或黄色条斑等病症，抽花后其花轴表面亦可发现有明显的条纹病症。病毒在晚香玉植株体内的存在，使得晚香玉植株生长缓慢，植株的每株花序数、座花率降低，花期缩短，花小，花色淡，商品价值大大降低。
侵染晚香玉的病毒寄主范围可能十分狭窄，人工接种了5科7种指示植物，结果表明，均未被晚香玉的病毒侵染，可能该病毒仅感染晚香玉一种植物；病毒粒体在晚香玉的花轴叶鞘、新生叶片、鳞叶含量较高，而鳞茎盘内携带的病毒粒体较为稀少；电镜观察侵染晚香玉的病毒为长丝状，病毒平均长度为750nm，病叶细胞质内见到针状内含体，扫描电镜下观察到风轮状内含体，因而从病毒分类上可以初步断定侵染晚香玉的是马铃薯Y病毒属的一种，并可能属于第二亚群。根据以上特征表明侵染黑龙江省晚香玉的病毒具体分类有待于进一步研究。
以东北农业大学园艺学院花卉专业提供之晚香玉的鳞茎0.15～0.3mm茎尖为外植体，以MS为基本培养基，添加蔗糖3％，琼脂0.8％，调pH值至5.7～5.8，通过激素配比试验，筛选出最佳的培养基组成。利用组织培养技术获得了晚香玉的无毒试管苗，结果表明：最佳诱芽培养基为MS+ IAA0.1mg／L +2-ip0.3mg／L，诱芽率为100％，最佳生根培养基为1／2MS+IBA1.0mg／L+NAA0.01mg／L,生根率为100％，利用此项组培技术，进行不同外植体大小、鳞茎热处理对脱毒效果的影响的研究表明在80℃高温条件下处理10min，然后切取0.3-0.5mm茎尖进行培养，脱毒率100％。
以东北农业大学园艺学院花卉专业提供之晚香玉的鳞茎茎尖为外植体，对晚香玉愈伤组织的诱导、分化、再生苗的生根和移栽进行了研究。结果表明：诱导愈伤组织最佳培养基为MS+2,4-D1.5 mg／l+BA0.5 mg／L，其出愈率达到90％；愈伤组织最佳分化培养基为MS+IAA0.1mg／L+2-ip0.3mg／L，分化率为87％，平均分化苗数6.7；再生植株生根壮苗的最佳培养基为1／2MS+IBA1.0mg／L+NAA0.01mg／L，生根率为100％，再生苗移栽后缓苗快，成活率100％。
晚香玉试管鳞茎微繁技术研究结果表明：接种4～6cm高的试管苗，在pH值为6.8，蔗糖浓度为90g／L，PP_(333)浓度为10mg／L的培养环境下最有利于晚香玉鳞茎的形成和膨大。试管鳞茎的直径为5～10mm时，移栽成活率高达97％～100％，并且鳞茎带叶和根有助于移栽成活。
通过以上研究，对晚香玉建立了有效的茎尖培养脱毒苗的快繁体系。
The incidence of single petal tuberose virus disease is about 45.95% ,which being cultured in Heilongjiang province.This is lower.But the incidence of double petal tuberose virus disease is higher about 97.3%.Almost all the infected plant have symptoms like mosaic leaf or yellow streak leaf. When floral axis growed we can observed obviouse streak symptoms in it.Virus existing in tuberose make the plant growthing slowly,reducing the number of inflorescence and the rate of flower production,cutting down the time of flowering.And the diseasing flower is small,the color is light.The value of tuberose commodty reduce greatly.
The range of tuberose hoest is very narrow. Inoculation 5 family 7species indicating plant against tuberose virus disease show that all the plant were not being infected.The virus maybe only infect tuberose.The content of granular virus is higher,which is existing in floral axis ,leaf sheath, new blade and bud scale.But in buib is lower.Electron microscopic observations of the naturally infected Tuberise revealed that there are fiexuous filamentous particles 750nm.Typical pinwheel type ,bundle are found in the cytoplasm of the mesophyll cell could be observed by ultrathin section of leaves.So tuberose virus can be concluded tentatively that is a kind of potyvirus and about belonging to the second group basing on above conclusion.The concret classify of tuberose virus in Heilongjiang province need to be identified continually.
Virus-free plants of tuberose 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 pH value is 5.7-5.8.A series of optimization experiment for cultural medium composition,concentration of phytohormones were investgated with a view to acclerate the propagation of virus 0 free plants.The result of virus elimination by meristem tip culture of purpie-skined tuberose commonly grown in North-east agriculture showed that the best medium shooting was MS+ LAA0.1 mg/1 +2-ip0.3 mg/L,the rate of taking shoot is 100%. And that for rooting was 1/2MS+IBA1.0 mg/L +NAA0.01 mg/L, the rate of taking root is 100%.Effect of virus-free culture with different meristem tips in size and heated -treated aer discussed. The result showed that pretreatment of cloces with 80 thermotheray for 10min,that obtained virus-free plants by using 0.3-0.5mm meristem tip.
This paper deals with studies on in vitro culture of meristem tip of Tuberose .including callus induction and differetiation, plantlet root development and transplantation. Results
showed :It was advantageous to callus induction on MS+2,4-D1.5 mg/1 +BA0.5 mg/L,the frequency of callus induction was 90%.MS+ LAA0.1 mg/L +2-ip0.3 mg/Lwas the best medium for callus differentition of which ratio was 87%,the maen number of plantlets per callus was 6.7.The best rooting medium was 1/2MS+IBA1.0 mg/L +NAA0.01 mg/L, 100%of the plantlets could develop their root.After the regenerated plantlets were transplanted to soil,they could adapt to environment.The survival ratio of transplanting reached 100%.
The researching of plantlet bulbing of Tuberose showed :It was advantageous to plantlet bulbing on the test tube of 4~6cm high,and pH6.8 , sucrose90g/L concentration of PP33310mg/L. Mean bulblet diameter was 5-10 mm made the ratio was 97~100%.Bulb with leaves and roots can make the ratio higher.
From these results,an effective procedure for mass propagation of virus-free tuberose by tip culture was established.
侵染晚香玉的病毒寄主范围可能十分狭窄，人工接种了5科7种指示植物，结果表明，均未被晚香玉的病毒侵染，可能该病毒仅感染晚香玉一种植物；病毒粒体在晚香玉的花轴叶鞘、新生叶片、鳞叶含量较高，而鳞茎盘内携带的病毒粒体较为稀少；电镜观察侵染晚香玉的病毒为长丝状，病毒平均长度为750nm，病叶细胞质内见到针状内含体，扫描电镜下观察到风轮状内含体，因而从病毒分类上可以初步断定侵染晚香玉的是马铃薯Y病毒属的一种，并可能属于第二亚群。根据以上特征表明侵染黑龙江省晚香玉的病毒具体分类有待于进一步研究。
以东北农业大学园艺学院花卉专业提供之晚香玉的鳞茎0.15～0.3mm茎尖为外植体，以MS为基本培养基，添加蔗糖3％，琼脂0.8％，调pH值至5.7～5.8，通过激素配比试验，筛选出最佳的培养基组成。利用组织培养技术获得了晚香玉的无毒试管苗，结果表明：最佳诱芽培养基为MS+ IAA0.1mg／L +2-ip0.3mg／L，诱芽率为100％，最佳生根培养基为1／2MS+IBA1.0mg／L+NAA0.01mg／L,生根率为100％，利用此项组培技术，进行不同外植体大小、鳞茎热处理对脱毒效果的影响的研究表明在80℃高温条件下处理10min，然后切取0.3-0.5mm茎尖进行培养，脱毒率100％。
以东北农业大学园艺学院花卉专业提供之晚香玉的鳞茎茎尖为外植体，对晚香玉愈伤组织的诱导、分化、再生苗的生根和移栽进行了研究。结果表明：诱导愈伤组织最佳培养基为MS+2,4-D1.5 mg／l+BA0.5 mg／L，其出愈率达到90％；愈伤组织最佳分化培养基为MS+IAA0.1mg／L+2-ip0.3mg／L，分化率为87％，平均分化苗数6.7；再生植株生根壮苗的最佳培养基为1／2MS+IBA1.0mg／L+NAA0.01mg／L，生根率为100％，再生苗移栽后缓苗快，成活率100％。
晚香玉试管鳞茎微繁技术研究结果表明：接种4～6cm高的试管苗，在pH值为6.8，蔗糖浓度为90g／L，PP_(333)浓度为10mg／L的培养环境下最有利于晚香玉鳞茎的形成和膨大。试管鳞茎的直径为5～10mm时，移栽成活率高达97％～100％，并且鳞茎带叶和根有助于移栽成活。
通过以上研究，对晚香玉建立了有效的茎尖培养脱毒苗的快繁体系。
The incidence of single petal tuberose virus disease is about 45.95% ,which being cultured in Heilongjiang province.This is lower.But the incidence of double petal tuberose virus disease is higher about 97.3%.Almost all the infected plant have symptoms like mosaic leaf or yellow streak leaf. When floral axis growed we can observed obviouse streak symptoms in it.Virus existing in tuberose make the plant growthing slowly,reducing the number of inflorescence and the rate of flower production,cutting down the time of flowering.And the diseasing flower is small,the color is light.The value of tuberose commodty reduce greatly.
The range of tuberose hoest is very narrow. Inoculation 5 family 7species indicating plant against tuberose virus disease show that all the plant were not being infected.The virus maybe only infect tuberose.The content of granular virus is higher,which is existing in floral axis ,leaf sheath, new blade and bud scale.But in buib is lower.Electron microscopic observations of the naturally infected Tuberise revealed that there are fiexuous filamentous particles 750nm.Typical pinwheel type ,bundle are found in the cytoplasm of the mesophyll cell could be observed by ultrathin section of leaves.So tuberose virus can be concluded tentatively that is a kind of potyvirus and about belonging to the second group basing on above conclusion.The concret classify of tuberose virus in Heilongjiang province need to be identified continually.
Virus-free plants of tuberose 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 pH value is 5.7-5.8.A series of optimization experiment for cultural medium composition,concentration of phytohormones were investgated with a view to acclerate the propagation of virus 0 free plants.The result of virus elimination by meristem tip culture of purpie-skined tuberose commonly grown in North-east agriculture showed that the best medium shooting was MS+ LAA0.1 mg/1 +2-ip0.3 mg/L,the rate of taking shoot is 100%. And that for rooting was 1/2MS+IBA1.0 mg/L +NAA0.01 mg/L, the rate of taking root is 100%.Effect of virus-free culture with different meristem tips in size and heated -treated aer discussed. The result showed that pretreatment of cloces with 80 thermotheray for 10min,that obtained virus-free plants by using 0.3-0.5mm meristem tip.
This paper deals with studies on in vitro culture of meristem tip of Tuberose .including callus induction and differetiation, plantlet root development and transplantation. Results
showed :It was advantageous to callus induction on MS+2,4-D1.5 mg/1 +BA0.5 mg/L,the frequency of callus induction was 90%.MS+ LAA0.1 mg/L +2-ip0.3 mg/Lwas the best medium for callus differentition of which ratio was 87%,the maen number of plantlets per callus was 6.7.The best rooting medium was 1/2MS+IBA1.0 mg/L +NAA0.01 mg/L, 100%of the plantlets could develop their root.After the regenerated plantlets were transplanted to soil,they could adapt to environment.The survival ratio of transplanting reached 100%.
The researching of plantlet bulbing of Tuberose showed :It was advantageous to plantlet bulbing on the test tube of 4~6cm high,and pH6.8 , sucrose90g/L concentration of PP33310mg/L. Mean bulblet diameter was 5-10 mm made the ratio was 97~100%.Bulb with leaves and roots can make the ratio higher.
From these results,an effective procedure for mass propagation of virus-free tuberose by tip culture was established.
引文
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55张淑娟等．洋桔梗F1无菌播种和试管苗的快速繁殖[J]．亚热带植物通，1996，25(2)：13-16
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65Bonga J M．Durzan DJ著。树木组合子培养．阙宁国，郭达初，李今田译．北京：中国林业出版社，1998，25～26，142～143，175
66Bos,L.,Huijberts. N.,and Maat, Z..Leek yellow stripe virus and relationships to onionyellow virus, characterization, eology and possible control.Neth. J.PI.path, 1978,84:185-204
67Bos,L.ylruses and virus disease of Allium species. Acta Horticulture, 1982,127:11-29
68Bravo-Almoacid,F.,Haim,L.,and mentaberry, A. Rappid immunological detection of potat viruses in pkant tissue squashes. Plant Dis. 1992, 76:574-578
69DEBERGHP, AITKETIE J,COHEND,et at Reconsideration of the term 'vitrification'as used in micropropagation[J].Plant Cell Tiss. Org. Cult, 1992,(30): 135-140
70DEBERGHP, HARBAOUI Y, LEMEUR R. Mass propagation of globe artichoke (Cynara scolyrnus):evalution of different hypotheses to overcome vitrification with special reference to water potential[J].Physiol Plant, 1981, (53): 181-187
71Fraenkel-Conrat,H..The viruses catalogue, characterization and classification. 1985,:107
72GASPAR T, KEVERS C, DEBERGH P, et al. Vitrification:morphological, physiological and ecological aspects [A]. In:Bonga J M,Durzan D J, eds. Celland Tissue Culture in Forestry[C].Dordrecht:Kluwer Academic Press Publ., 1987,1:152-166
73Gibbs,A.,and Harrison,B. 1976.Plant Virology-The Principles. Edward Arnold Ltd. London. :292
74Haissig B E, et al. Trends in the use of tissue culture in forest improvement[J]. Bio/Technology, 1987,5: 52.
75Haissig B E, et al. Trends in the use of tissue culture in forest improvement[J].Bio\Technology, 1987,5:52
76Halepyati AS, Effect of moisture stress and population densities on tuberose[J].Journal of
Maharashtra Agricultural universities, 1995, (20):2,205-208
77Hsu.h.t.,Kim,J.Y.,AND Lawson, R.H.Purification of lilysymptomless carlavirus and detection of the virus in lilies.plant Dis. 1995,79:912-916
78Kee-Youep Paek,et al. Advances in development biology and biotechnology of higher plants[A].Tissue Culture[M].Published Korea Soc, 1993.38
79Lee,Y,W.,Yamazaki,T.,And Inouye,T..Two elongated viruses in garlic, garlic latent virus and garlic mosaic virus.Nann. Phytoph. Soc. Japan, 1979,45:727-734
80Meria. Ziv:Inhanced shoot and cormlet proliferation in liquid cultured gladiolus buds by growth retardants. Plant cell Tissue and culture, 1989, 17:101-110
81Pena-Iglesian,A..Characterization of Spanish garlic viruses and their elimination by in virtro shoot apex culture. Aaacta Horticulture,1982,127:183-193
82PREECE J E, SUTTER E G. Acclimation of Micropropation-Technology and pplication[Z]. Dordrecht:Kluwer Academic Press Publ,19990:71-93
83Shen ,T.M.,Shen,R.S.,and Huang ,K.L..Studies on the flowering of tuberose(Polianthes tuberosa L.).Journal of the Chinese Society for Horticultural Science. 1991, 37(1):10-20
84Singh K P. Improved production technologies for tuberose[J]. Agricultural-Reviews-Karnal, 1995,16(3): 141-166
85Stein, A., Levy, S., and Loebenstein, G. Detection of viruse in gladioli corms. Acta Hortic. 1988, 234:275-280
86Steinitz B, lilien-Kipris H. Control of precocious gladiolus coem and cormel formation in tissue culrure. J lantPhysiol,1989,135:495-500
87Van der Vlugt,C.I.M.,Derk,A.F.L.M.,Boonekamp,P.M.,and Goldbah,R.W. Development of test-procedures for the detection of iris severe mosaic virus in secondarily and primarly-infected bulbs. Acta Hortic. 1994, 377:197-198
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65Bonga J M．Durzan DJ著。树木组合子培养．阙宁国，郭达初，李今田译．北京：中国林业出版社，1998，25～26，142～143，175
66Bos,L.,Huijberts. N.,and Maat, Z..Leek yellow stripe virus and relationships to onionyellow virus, characterization, eology and possible control.Neth. J.PI.path, 1978,84:185-204
67Bos,L.ylruses and virus disease of Allium species. Acta Horticulture, 1982,127:11-29
68Bravo-Almoacid,F.,Haim,L.,and mentaberry, A. Rappid immunological detection of potat viruses in pkant tissue squashes. Plant Dis. 1992, 76:574-578
69DEBERGHP, AITKETIE J,COHEND,et at Reconsideration of the term 'vitrification'as used in micropropagation[J].Plant Cell Tiss. Org. Cult, 1992,(30): 135-140
70DEBERGHP, HARBAOUI Y, LEMEUR R. Mass propagation of globe artichoke (Cynara scolyrnus):evalution of different hypotheses to overcome vitrification with special reference to water potential[J].Physiol Plant, 1981, (53): 181-187
71Fraenkel-Conrat,H..The viruses catalogue, characterization and classification. 1985,:107
72GASPAR T, KEVERS C, DEBERGH P, et al. Vitrification:morphological, physiological and ecological aspects [A]. In:Bonga J M,Durzan D J, eds. Celland Tissue Culture in Forestry[C].Dordrecht:Kluwer Academic Press Publ., 1987,1:152-166
73Gibbs,A.,and Harrison,B. 1976.Plant Virology-The Principles. Edward Arnold Ltd. London. :292
74Haissig B E, et al. Trends in the use of tissue culture in forest improvement[J]. Bio/Technology, 1987,5: 52.
75Haissig B E, et al. Trends in the use of tissue culture in forest improvement[J].Bio\Technology, 1987,5:52
76Halepyati AS, Effect of moisture stress and population densities on tuberose[J].Journal of
Maharashtra Agricultural universities, 1995, (20):2,205-208
77Hsu.h.t.,Kim,J.Y.,AND Lawson, R.H.Purification of lilysymptomless carlavirus and detection of the virus in lilies.plant Dis. 1995,79:912-916
78Kee-Youep Paek,et al. Advances in development biology and biotechnology of higher plants[A].Tissue Culture[M].Published Korea Soc, 1993.38
79Lee,Y,W.,Yamazaki,T.,And Inouye,T..Two elongated viruses in garlic, garlic latent virus and garlic mosaic virus.Nann. Phytoph. Soc. Japan, 1979,45:727-734
80Meria. Ziv:Inhanced shoot and cormlet proliferation in liquid cultured gladiolus buds by growth retardants. Plant cell Tissue and culture, 1989, 17:101-110
81Pena-Iglesian,A..Characterization of Spanish garlic viruses and their elimination by in virtro shoot apex culture. Aaacta Horticulture,1982,127:183-193
82PREECE J E, SUTTER E G. Acclimation of Micropropation-Technology and pplication[Z]. Dordrecht:Kluwer Academic Press Publ,19990:71-93
83Shen ,T.M.,Shen,R.S.,and Huang ,K.L..Studies on the flowering of tuberose(Polianthes tuberosa L.).Journal of the Chinese Society for Horticultural Science. 1991, 37(1):10-20
84Singh K P. Improved production technologies for tuberose[J]. Agricultural-Reviews-Karnal, 1995,16(3): 141-166
85Stein, A., Levy, S., and Loebenstein, G. Detection of viruse in gladioli corms. Acta Hortic. 1988, 234:275-280
86Steinitz B, lilien-Kipris H. Control of precocious gladiolus coem and cormel formation in tissue culrure. J lantPhysiol,1989,135:495-500
87Van der Vlugt,C.I.M.,Derk,A.F.L.M.,Boonekamp,P.M.,and Goldbah,R.W. Development of test-procedures for the detection of iris severe mosaic virus in secondarily and primarly-infected bulbs. Acta Hortic. 1994, 377:197-198
88ZIV M.Vitrification:morphological and physiological disorders of in vitro plants[A]In:debergh P C, Zimmerman R H, eds. Micorpropagation-Technology and Application[Z].Dordrecht:Kluwer Academic Press Publ, 1991:45-69