大蒜茎尖脱毒体系的建立与病毒电镜检测分析
摘要
大蒜(Allium sativum L.)是一种重要的蔬菜作物,生产中主要靠鳞茎进行无性繁殖。大蒜在长期无性繁殖过程中,易感染和积累多种病毒,且广泛传播,造成种性退化,品质与产量降低。应用大蒜茎尖分生组织并结合热处理是脱除大蒜病毒的行之有效的方法,可以有效脱除大多数病毒,大幅度提高大蒜的品质与产量。但大蒜茎尖培养难度较大,繁殖系数较低,病毒鉴定困难,脱毒良种生产成本较高,成为大蒜脱毒良种培育与生产应用的瓶颈问题。
本研究以茎尖分生组织培养结合热处理的方法脱除大蒜病毒,建立大蒜茎尖脱毒培养体系,并以新鲜的或低温贮藏的蒜薹为外植体,研究了贮藏时间和植物生长激素对花苞培养诱导形成气生鳞茎的影响,以及气生鳞茎大小、处理温度和植物激素配比对气生鳞茎的萌发率的影响。利用扫描电镜技术检测大蒜脱病毒前后培养组织所含病毒情况。取得以下研究结果:
(1)利用成县迟蒜和鲁蒜王Ⅱ号两个不同品种的鳞茎经行热处理和茎尖脱毒研究,建立了大蒜茎尖脱毒培养体系。结果表明,在MS+0.1 mg/L NAA+0.5 mg/L6-BA的培养基上, 0.3mm左右的大蒜茎尖有较高的萌芽率和试管鳞茎形成率,生长势也较强,萌芽率和试管鳞茎形成率可达到97.4%和87.2%;经试验比较,在37℃下热处理30d后,大蒜茎尖有较高的鳞茎形成率。
(2)大蒜花苞可用作离体条件下的外植体材料诱导形成气生鳞茎,但是受到贮藏时间的影响。未经贮藏的外植体在MS+NAA 0.15 mg/L+6-BA 0.3 mg/L培养基激素配比适宜,能诱导较多的气生鳞茎;经4℃低温贮藏60 d后,大蒜花苞在MS+NAA 0.1 mg/L+ABA 1.0 mg/L激素配比培养基上气生鳞茎诱导率可达95.0%。大蒜气生鳞茎萌发受到其个体大小的影响,较小的气生鳞茎能迅速且萌发率高,较大的气生鳞茎则萌发率低。气生鳞茎在MS+NAA 0.05 mg/L+6-BA 0.1 mg/L培养基激素配比较适宜,能诱导较多的气生鳞茎萌发。
(3)扫描电镜检测结果表明,未进行茎尖培养前,成县迟蒜被马铃薯Y病毒属、青葱潜隐病毒属、大蒜潜隐病毒属、麝香石竹潜隐病毒属的病毒侵染;鲁蒜王Ⅱ号被麝香石竹潜隐病毒属、马铃薯Y病毒属、一些长线病毒侵染;在经过茎尖脱病毒后,在两个品种中只检测到少量麝香石竹潜隐病毒属和马铃薯Y病毒属。表明在本研究范围内,大蒜茎尖培养配合热处理可对大蒜病毒进行有效脱除,脱毒率达到60%以上。
Garlic (Allium sativum L.) is an important vegetable crops, the production of asexual reproduction mainly by the bulb. Garlic in the long process of asexual reproduction , accumulation of susceptible viruses and widespread, resulting in degradation of caste, quality and yield. Garlic apical meristem removal combined with heat treatment is an effective method of garlic virus, the virus can be effectively removed most of the substantial increase in the quality and yield of garlic. However, difficult the garlic shoot tip culture propagation coefficient low, virus identification difficulties, virus-free seed production costs high, a virus-free garlic seed cultivation and production of application bottlenecks.
This research shoot apical meristem culture of garlic with heat treatment approach removal of viruses,and to establish the garlic shoot tips virus-free culture system. And substitute with fresh or low temperature storage of garlic sprouts as explants to study the storage time and culture of plant growth hormones induce the formation of inflorescence axis of bulbils, and the size of bulbils, temperature and plant hormone on aerial germination rate of the bulbs. Detected by scanning electron microscopy before and after tissue culture of garlic removal virus contained in the virus situation.
(1) Two species of garlic bulbs by the line combination of heat treatment and detoxification of tip growing point.The results showed that MS +0.1 mg / L NAA +0.5 mg/L6-BA garlic shoot tips on medium had a higher germination rate and in vitro bulb formation rate, but also a strong growth potential, germination rate and in vitro bulb formation rate up to 97.4% and 87.2%; 30d at 37℃heat treatment after the bulb of garlic shoot tips have a higher formation rate; cut the size of 0.3 ~ 0.5mm tip, the tip have a higher survival rate, bulb formation rate and growth potential.
(2)The inflorescence of garlic can be used as an explant in vitro to formed bulblets. After low temperature storage, formation rate of bulblets is relatively low. Formation of bulblets germinated by the impact of bulblets size. By MS as medium,with no storage and storage30,60,90and120d, respectively, the inflorescences of garlic were used as explants for tissue culture.The results indicated that the formation rate of bulblets could reach 95.0% under the interactions treatments of MS+NAA 0.1 mg/L+ABA 1.0 mg/L,after 60 d storage at 4℃. Small bulblets can germinate rapidly and theirs shooting rate is high reached 77.9%,but big bulblets come to the opposite.The MS+ NAA 0.05 mg/L+6-BA 0.1 mg/L medium is appropriate proportion,which can induce more bulblet shooting
(3)In order to provide theroretical guidance for screening no virus plants and application, electron microscope technology is used to detect the virus of the Late garlic from Gansu and King of garlic Lu from Shandong.The results showed in the Late garlic from Gansu it had Potyvirus,Shallot latent virus(ShLY), Garlic common latent virus and Carlavirus;in King of garlic Lu from Shandong garlic it had Shallot latent virus(ShLY), Potyvirus and many different long length virus. That within the scope of this study, treatment with garlic shoot tip culture of garlic can be effective virus removal, virus-free rate of 60% or more.
本研究以茎尖分生组织培养结合热处理的方法脱除大蒜病毒,建立大蒜茎尖脱毒培养体系,并以新鲜的或低温贮藏的蒜薹为外植体,研究了贮藏时间和植物生长激素对花苞培养诱导形成气生鳞茎的影响,以及气生鳞茎大小、处理温度和植物激素配比对气生鳞茎的萌发率的影响。利用扫描电镜技术检测大蒜脱病毒前后培养组织所含病毒情况。取得以下研究结果:
(1)利用成县迟蒜和鲁蒜王Ⅱ号两个不同品种的鳞茎经行热处理和茎尖脱毒研究,建立了大蒜茎尖脱毒培养体系。结果表明,在MS+0.1 mg/L NAA+0.5 mg/L6-BA的培养基上, 0.3mm左右的大蒜茎尖有较高的萌芽率和试管鳞茎形成率,生长势也较强,萌芽率和试管鳞茎形成率可达到97.4%和87.2%;经试验比较,在37℃下热处理30d后,大蒜茎尖有较高的鳞茎形成率。
(2)大蒜花苞可用作离体条件下的外植体材料诱导形成气生鳞茎,但是受到贮藏时间的影响。未经贮藏的外植体在MS+NAA 0.15 mg/L+6-BA 0.3 mg/L培养基激素配比适宜,能诱导较多的气生鳞茎;经4℃低温贮藏60 d后,大蒜花苞在MS+NAA 0.1 mg/L+ABA 1.0 mg/L激素配比培养基上气生鳞茎诱导率可达95.0%。大蒜气生鳞茎萌发受到其个体大小的影响,较小的气生鳞茎能迅速且萌发率高,较大的气生鳞茎则萌发率低。气生鳞茎在MS+NAA 0.05 mg/L+6-BA 0.1 mg/L培养基激素配比较适宜,能诱导较多的气生鳞茎萌发。
(3)扫描电镜检测结果表明,未进行茎尖培养前,成县迟蒜被马铃薯Y病毒属、青葱潜隐病毒属、大蒜潜隐病毒属、麝香石竹潜隐病毒属的病毒侵染;鲁蒜王Ⅱ号被麝香石竹潜隐病毒属、马铃薯Y病毒属、一些长线病毒侵染;在经过茎尖脱病毒后,在两个品种中只检测到少量麝香石竹潜隐病毒属和马铃薯Y病毒属。表明在本研究范围内,大蒜茎尖培养配合热处理可对大蒜病毒进行有效脱除,脱毒率达到60%以上。
Garlic (Allium sativum L.) is an important vegetable crops, the production of asexual reproduction mainly by the bulb. Garlic in the long process of asexual reproduction , accumulation of susceptible viruses and widespread, resulting in degradation of caste, quality and yield. Garlic apical meristem removal combined with heat treatment is an effective method of garlic virus, the virus can be effectively removed most of the substantial increase in the quality and yield of garlic. However, difficult the garlic shoot tip culture propagation coefficient low, virus identification difficulties, virus-free seed production costs high, a virus-free garlic seed cultivation and production of application bottlenecks.
This research shoot apical meristem culture of garlic with heat treatment approach removal of viruses,and to establish the garlic shoot tips virus-free culture system. And substitute with fresh or low temperature storage of garlic sprouts as explants to study the storage time and culture of plant growth hormones induce the formation of inflorescence axis of bulbils, and the size of bulbils, temperature and plant hormone on aerial germination rate of the bulbs. Detected by scanning electron microscopy before and after tissue culture of garlic removal virus contained in the virus situation.
(1) Two species of garlic bulbs by the line combination of heat treatment and detoxification of tip growing point.The results showed that MS +0.1 mg / L NAA +0.5 mg/L6-BA garlic shoot tips on medium had a higher germination rate and in vitro bulb formation rate, but also a strong growth potential, germination rate and in vitro bulb formation rate up to 97.4% and 87.2%; 30d at 37℃heat treatment after the bulb of garlic shoot tips have a higher formation rate; cut the size of 0.3 ~ 0.5mm tip, the tip have a higher survival rate, bulb formation rate and growth potential.
(2)The inflorescence of garlic can be used as an explant in vitro to formed bulblets. After low temperature storage, formation rate of bulblets is relatively low. Formation of bulblets germinated by the impact of bulblets size. By MS as medium,with no storage and storage30,60,90and120d, respectively, the inflorescences of garlic were used as explants for tissue culture.The results indicated that the formation rate of bulblets could reach 95.0% under the interactions treatments of MS+NAA 0.1 mg/L+ABA 1.0 mg/L,after 60 d storage at 4℃. Small bulblets can germinate rapidly and theirs shooting rate is high reached 77.9%,but big bulblets come to the opposite.The MS+ NAA 0.05 mg/L+6-BA 0.1 mg/L medium is appropriate proportion,which can induce more bulblet shooting
(3)In order to provide theroretical guidance for screening no virus plants and application, electron microscope technology is used to detect the virus of the Late garlic from Gansu and King of garlic Lu from Shandong.The results showed in the Late garlic from Gansu it had Potyvirus,Shallot latent virus(ShLY), Garlic common latent virus and Carlavirus;in King of garlic Lu from Shandong garlic it had Shallot latent virus(ShLY), Potyvirus and many different long length virus. That within the scope of this study, treatment with garlic shoot tip culture of garlic can be effective virus removal, virus-free rate of 60% or more.
引文
[1] Abo E L,Nil M M.Organogenesis and embryogenesis in callus of garlic(Allium sativum L.)[J].Plant Science Letter,1977,(9):259-264
[2] Amasino RM. Vernalization and flowering time [J].Curr Opin Biotech, 2005,16: 154-158
[3] Ayabe M,Sumi S.Establishment of novel tissue culture method,stem-disc culture, and its practical application to micropropagation of garlic(Allium sativum L.)[J].Plant Cell Reports,1998,17:773-779
[4] Baker R,etal.ln:kruse PF Jr,Patterson M K Jr.eds. Tissue Culture: Methure: Methods and Application[J].New York:Academie Press,1973,735-739
[5] Ball E M and Barke M K.leaf-dip serology for electron microscope identification of plant viruses[J].Virology,1968,36:152-155
[6] Burdon R H.Superoxide and hydrogen peroxide in relation to mammalian cell proliferation[J].Free Radical Biology and Medicine,1995,18:775-779
[7] Bertaeeini A,Botti S,Tabanelli D,et al.Micropropagation and establishment of mite borne virus free garlic(Allium sativum L.)[J].Acta Horticulturae,2004,6(31):201-206
[8] Camara ,M Pasqual, JEBP Pinto. Obtaining plants from garlic(Allium sativum L.)cultivars Chinese and Chonan using in vitro cultured meristems[J].Cienciae Pratica. 1989,13(1):97~101
[9] Chen J,Zheng H Y,Antoniw J F,Adams M J,Chen J P,Lin L.Detection and classification of allexiviruses from garlic in China [J].Archives of Virotogy,2004,149,435-445
[10] Choi S L,Paek K Y,Kwun K C.Effect of explants source and shoot trimming on shoot multiplication and bulb formation of garlic in vitro[J].Jounal of the Korean Society for Horticultural Science,1985,26(4):304-312
[11] Conci V ,Nome S F,Milne R G.Filamentous viruses of garlic in argentina [J] . Plant Dis,1992, 76:594-596
[12] Dare K C,Oberley T D,Mouse K E,et al . Expression of manganese superoxide dismutase Promote cellular differentiation[J].Free Radical Biology and Medicine,1994,16:275
[13] Derrick K S.Qauntitative assay for plant viruses using serologically specific electron microscopy [J].Virology,1973,56: 652
[14] Du Y Q, Zhu J ZH,Shen W P.Study on virus elimination technology of garlic in Jiading by shoot tip culture[J].Acta Agriculturae Shanghai,2004,20(l):9-12
[15] Haque M S,Wada T,Hattorik.Garlic roots for micropropagation through in vitro bulblet formation[J]. Aeta Horticulturae,2000,No.520,45-52
[16] Hatta T,Franclz R L B.Cytopathic structures as-sociated with tonoplasts of plant cells infected with cucumber mosaic and tomato aspermy virus[J].Journal of General Virology ,1981,53: 343-347
[17] Hernandez PR,Prado O,Gil DV.1994.Production of virus-free garlic(Allium sativum L.)bymeristem culture[J].Centro Agricola.21(1):76~84
[18] Hus H T, Aebig J. Enzyme-linked immuunosorbent assay (ELISA)of plant viruses using protein A-enzyme conjugate and F(ab’)of immunglobin G(IgG)[J]. Phytopathology ,1982 ,74:708
[19] Kawarabayashi W,Asahira T.In vitro Multiplication of virus-free bublbs of Lilies [J].Japan Soc Hort Sci,1989,58:195-209.
[20] Khan N,Alam M S,Nath U K.In vitro regeneration of garlic through callus culture[J].Journal of Biological Sciences,2004,4(2):189-191
[21] Kim E K, Hahn E J,Murthy H N,et al.Enhanced shoot and bulblet proliferation of garlic(Allium sativum L.)in bioreactor systems[J].Journal of Horticultural Science and Biotechnology, 2004,79(5):818-822
[22] Kinard G R,Scott S W,Barnett O W. Detection of apple chlorotic leaf spot and apple stem grooving viruses using RT-PCR[J]. Plant Disease ,1996, 80(6): 616-621
[23] Komissarov V A.Bulblet culture of garlic.[J].Kartofel Ovoshchi,1997,(2):23-24
[24] Kudou R,Fujime Y,Amimoto K. Effects of plant growth regulators and sampling positions on organ formation of garlic.Technical Bulletin of the Faculty of Agriculture[J],Kagawa University,1995,47(l):15-22
[25] Lubraco G,Schubert A,Previati A. Micropropagation and mycorrhization of Allium sativum[J].Acta Horticulturae,2000,(530):339-343
[26] Ma Y,Wang H L,Zhang C J .High rate of virus free plantlet regeneration via garlic scrapes tip culture[J].Plant Cell Reports,1994,14(l):65-68
[27] Matsubars S, Chen D. In vitro production of garlic plants and field acclimatization [J]. Scientia Horticulturae,1989,4: 677-679
[28] MD Late Submission Abstracts in Vitro Biology Meeting[C].2005 meeting of the society for in vitro biology, June 5-7, Baltimore, 2005. 3028.
[29] Mohamed Y Y,Barringer S A, Splittstoesser W E.In vitro bulb production from Allium spp.[J].In Vitro Cellular and Developmental Biology Plant,1995,31(1):51-52
[30] Moriconi D N,Conci V C,Nome S F.In vitro plantlet regeneration from callus in garlic (Allium sativum L.)[J].Phytopathlogy Buenos Aires,1989,49(1-2):97-103
[31] Nagakubo T, Nagasawa A,Ohkawa H. Micropropagation of garlic through in vitro bulblet formation [J]. Plant Cell Tissue and Organ Culture,1993,32(2): 175-183
[32] Navarro L.Citrus shoot-tip grafting in vitro (STG) and its applications:a review Proc[J].Int. Soc. Citriculture,1981:l:452-456.
[33] Navarro L,Roistaeher N C,Murshige T.Improvement of shoot-tip grafting in vitro for virus-free citrus[J].Amer.Soc.Hort.Sci.,1975,100(5):471-479.
[34] Osaws K, Sugawara H. Studies on techniques of tissue culture一I .Experiment on the method of acclimatization of in vitro plantlets of vegetables [J]. Bulletin of Developmental Breeding Vegetables and Organic Crops,1980,7: 22-25
[35] Ploaie P G.Use of immune electron microscopy o detect viruses in plants regenerated from meristem tip cultures[J].Buletinul de Protectia Plantlor,1984
[36] Quak.F.,Applied and Fundamental Aspects of Plant Cell,Tissue and organ Culture.Ed.By J.Reinert andY.P.S.Bajaj,Springer-Verlag, Berlin,PP.1977:596-615
[37] Robledo P A, Villalobos A M , Jofre G E .Efficient plant regeneration of garlic (Allium sarivum L.)by root tip culture[J].InVitro Cellular and Developmental Biology Plant,2000,36(5):416-419
[38] Seabrook J A.In vitro propagation and bulb formation of garlic[J].Canadian Journal of Plant Science,1994,74(1):155-158
[39] Spiegel S,Scot S W,Bowman V,et al. Improved detection of Prunus necrotic ringspot virus by the polymerase chain reaction[J]. European Journal of Plant Pathology,1996,102(7) : 681-685
[40] Sumi S,Matsumi T,Tsuneyoshi T .Complete nucleotide sequences of garlic viruses A and C,members of the newly ratified genus Allexivirus[J].Archives of Virology,1999,144:1819-1826
[41] Suh S K,Park H G.Rapid multiplication through immature bulbil cultures of garlic[J].Journal of the Korean Society for Horticultural Sciencf,1993,34(3):173-178
[42] Takagi H, Qu Y.Effect of light quality, photoperiod and cold treatment on in vitro bulbing of garlic shoot tip [J]. Acta Horticulturae,1995,(393): 181-188
[43] Ucman R, Zel J, Ravnikar M. Thennotherapy in virus elimination from garlic: Influences on shoot multiplication from meristems and bulb formation in vitro[J]. Scientia Horticulturae,1998, 73(4):193-202
[44] Van Dijk P.Carlavirus isolates from cultivated Alliumspecies represent three viruses.Neth [J]Plant Pathol,1993,99: 233-257
[45] Van Dijk P.Survey and characterization of poty viruses and their strains of Allium species[J].Neth.J.Plant Pathil.,1993,99: 38-41
[46] Verbeek M,van Dijk P,van Well PMA.Efficieney of eradication of four viruses from garlie (Allium sativum L.) by meristem tip culture [J].European Journal of Plant Pathology,1995,101:3,231-239
[47] Walkey D.G.A, etal. 1987. Agronomic evaluation of virus-free and virus-infected garlic(Allium sativum L.)[J].J. Hort. sci.64(1):53-60
[48] Yasseen M Y, Walter E S, Richard E L. In vitro shoot proliferation of sets from garlic and shallot [J]. Plant Cell Tissue and Organ Culture,1994, 36: 243-247
[49]昌祖慧.大蒜病毒病及其防治方法[J]湖北植保,1995,(5): 20
[50]陈典.大蒜脱毒种苗培育技术研究[J].北方园艺,1997, (2): 29-30.
[51]陈炯,陈剑平.Allexivirus属的一个新成员—大蒜病毒E的基因组全序列测定和系统树分析.[J]科学通报,2001, (40):1463-1468
[52]陈建平.胶体金免疫电镜技术检测和鉴定病汁液中不同形态的植物病毒[J].植物生理学报,1993,23(2):169-174
[53]陈青奇,陈典,张海霞.大蒜育种研究现状[J].北方园艺,2006(2):40-41
[54]陈世儒,黄菊辉.大蒜离体快繁及脱毒[J].园艺学报,1991,3(3):133-137
[55]崔凯荣,邢更生,周功克.体细胞胚发生的生化基础[[J].生命科学,2001, 13(1): 28-33
[56]崔荣昌,等.大蒜花叶病原的鉴定和茎尖脱毒效果[J].中国蔬菜,1992,4:10-14
[57]崔月花.张彪.高红明.等.唐营蒲茎尖培养脱病毒的研究[J].江苏农业研究,2000,21(4):88-89.
[58]丁辛顺.脱毒大蒜苗的生产检测技术[J].上海农业学报,1998,4(1):23~28
[59]高山林,金雍安,蔡朝晖.大蒜分生组织培养脱病毒和快速繁殖技术[J].植物资源与环境学报,2000, 9(3): 5-18
[60]高述民,陆帼一.大蒜体细胞胚胎发生研究进展[J].植物学通报,2000,17(4): 338-344
[61]杭玲,潘颖南,黄卓忠.草墓甸旬茎尖组织培养与脱毒苗生产田[J].广西林业科学,1999,6:319-320
[62]洪健,李德葆,周雪平.植物病毒分类图谱(第一版)[M].北京:科学出版社,2001
[63]焦奎,张书圣.伏安酶联免疫分析法及其在植物血清学检测技术中的应用[J].化学通报,2000,(10): 50-55
[64]兰平,李文凤,朱水芳.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(自然科学版),2001,3:1-4.
[65]李亚男,陈大清.脱落酸和赤霉素对大蒜某些生理特性的影响[J].湖北农学院学报,16(2):93-97.
[66]李昌华,李小川,赵美华等.大蒜茎尖脱毒技术及组织培养研究[J].华北农学报. 1995,10(3):20-25
[67]李小川,赵美华.大蒜鳞芽生长点离体培养诱导小鳞茎的形成[J].山西农业科学,1996, 24(3):59-60
[68]刘华.环境条件对马铃薯病毒生物测定结果的影响[J].山西农业科学,2003,31(2): 72-74
[69]刘高琼,李式军,张学平.温光条件对离体大蒜鳞茎形成和内源激素变化的效应[J].园艺学报,1997,4(2):165-16
[70]陆关成,李方.余姚白蒜组织培养脱毒的研究.浙江农业学报,1991,3(3):133-137
[71]鲁宇文.侵染大蒜的线状植物病毒的外壳蛋白基因原核表达、抗血清制备及检测应用[M].浙江大学2006年硕士论文.
[72]栾雨时,奕非时,崔喜波.大蒜茎尖培养脱毒效果[J].北方园艺,1996(4):37
[73]栾非时,陈典,陈友.脱毒大蒜花原始体培养增殖技术的研究[J].中国蔬菜,1995. (3):4-6.
[74]刘华.环境条件对马铃薯病毒生物测定结果的影响[J].山西农业科学,2003,31(2): 72-74
[75]李向东,于晓庆,古勤生等.马铃薯Y病毒属病毒基因功能研究进展[J].山东科学,2006,(3):1-6
[76]马筠.植物病毒鉴定检测方法的研究进展[J].世界农业,2003,292: 50-51
[77]孟振农,陈永,赵启韬等.大蒜花序中不同繁殖器官的形态发生[J].山东大学学报(自然科学版),1997,32(3):332-336
[78]牛淑妍,焦奎,张成良.联苯胺-H202-HRP伏安酶联免疫分析体系测定植物病毒TMV和TRSV[J].分析科学学报,1998,17 (3):12-14
[79]彭宏梅,李月秋,张仲凯等.应用电镜技术研究大理州烟草苗期病毒病的发生情况.[J]云南农业大学学报,2000,15(1):71-72
[80]彭于发,王慧敏,彭有良.植物病理学研究[M].北京:中国农业出版社,1997.9-13.
[81]齐放军,贾敬芬,李继胜.抗氧化剂对首楷原生质体早期培养的影响[J].实验生物学报,1994,27(l):1-9
[82]佘建明,丁犁平,陆维忠等.大蒜体细胞诱导再生植株和小鳞茎及其移栽试验[J].江苏农业学报,1992,8(4):46-47
[83]施曼玲,周雪平.植物病毒的诊断技术[J].微生物学通报,2000,27(2): 149-151
[84]宋瑞琳,吴如健,柯冲.茎尖嫁接脱除柑桔主要病原的研究[J].植物病理学报,1999,29(3):275-279.
[85]宋元林,唐顺明等.大蒜、洋葱、葱、韭葱栽培新技术[M].北京:中国农业出版社. 1999.1~43
[86]土杰,张大伟,范卫红等.侵染西瓜的烟草花叶病毒(TMV)的生物学种类鉴定[J].安徽农业科学,2003, 31(6):972-974
[87]王文和,邓馨,胡文玉等.草幕叶片愈伤组织形成及再生芽分化的组织学研究[J].沈阳农业大学学报1999,30(4):430-433.
[88]王忠.植物生理学[M].北京:中国农业出版社,2000:415-417
[89]邢更妹,李杉,崔凯荣等.植物体细胞胚发生中某些机理探讨[J].自然科学进展,2000,10(8):684-692
[90]熊正琴,李式军,刘高琼等.大蒜花序轴离体培养的研究[J].南京农业大学学报,2000,23(3):25-28
[91]徐洁.指示植物鉴定马铃薯病毒技术的研究与应用[J].中国马铃薯,2002,(2) :73-75
[92]徐培文,孙慧生,孙瑞杰.脱毒大蒜速繁途径探讨和良繁体系的建立.中国蔬菜,1993,5:9-13
[93]徐培文,孙慧生,孙瑞杰等.大蒜茎尖培养脱毒及增产效果的研究[J].山东农业科学,1984,6:6-10
[94]徐培文,孙惠生,孙瑞杰等.大蒜脱毒技术及应用研究[J].中国农业科学,1998,31(2):92-94
[95]薛万新,陆帼一.大蒜蒜瓣离体繁殖研究[J].西北农业学报,1994, 3(3): 62-66
[96]徐品三,栗雨时,刘纪文等.百合不定芽培养脱毒种球生产的研究[J].植物学通报,2003,20(3):313-318.
[97]徐平东,李梅,林奇英等.应用A蛋白夹心酶联免疫吸附法鉴定黄瓜花叶病毒血清组[J].福建农业大学学报,1997,26 (1): 64-69
[98]徐平珍,刘涛,杨莹等.脱落酸在植物花发育过程中的作用[J].云南植物研究,2007,29(2),215-222.
[99]徐启江,陈典,张云修.分孽洋葱茎尖培养脱毒苗技术研究[J].西北农林科技大学学报,2002,30(2):102-106.
[100]薛光荣,杨振英,洪霓等.茎尖培养等处理脱除梨病毒的技术研究[J].中国果树,1996(3):9-11
[101]杨乃博.大蒜全展叶愈伤组织的诱导和植株再生[J].植物生理学通讯,1992(18):47-48
[102]杨有权:周清海:杨娟.大蒜瞒类害虫的研究[J]长江蔬菜1998,(3) :17-21
[103]杨有权,杨素真,王学国.大蒜病毒病及其防治[J]长江蔬菜.1997 ( 9) : 17-18
[104]于德才.大蒜茎尖脱毒及快繁研究[J].北方园艺,2005, (6):84-85
[105]远彤,李坷,闰文斌等.大蒜花芽分化的细胞组织学研究[J].河南农业大学学报,1996,30(2): 186-190
[106]运广荣主编.中国蔬菜实用新技术大全(北方蔬菜卷)[M].北京:科学技术出版社. 2004.515-518
[107]魏宁生,吴云峰.大蒜病毒病原的鉴定及组培脱毒研究[J].西北农业大学学报,1992,20(1): 76-81
[108]赵军良.植物茎尖培养与无毒种苗生产[J].北方园艺,1995,6:10-12.
[109]赵顺庆,李鹏飞,范怀忠.花叶病大蒜的茎端组织脱毒培养.华南农业大学学报,1987,8(4):l-8
[110]张昌伟,侯喜林,袁建玉等.太仓大蒜根尖离体培养直接诱导不定芽及其试管鳞茎的形成[J].植物生理学通讯,2004(2):167-170
[111]张德玉,刘成林,尚化芳等.大蒜病毒病的发生及综合防治技术[J].蔬菜2006, ( 8):27
[112]张明厚,魏培文,朱俊华.大蒜茎尖组培苗的检测[J].东北农业大学学报,1999,30(2):105-110
[113]张素芝,李纪蓉.一种新型的大蒜快繁体系——花苞不定芽再生体系的建立[J].山东农业大学学报(自然科学版),2007,38(2):159-162
[114]张素芝,李纪蓉.影响大蒜气生鳞茎萌发的因素分析[J].四川农业大学学报,2006,24(2):148-151
[115]张素芝,李纪蓉.植物激素配比对大蒜茎盘愈伤组织再生体系的影响[J].种子,2006(6):38-40.
[116]张明厚,魏培文,朱俊华等.大蒜茎尖组培苗的检测[J]东北农业大学学报,1999, 30(2), 105-110
[117]张仲凯,李毅.云南植物病毒[M].北京:科学出版社,2001
[118]郑海柔.大蒜试管苗中鳞茎的诱导[J].上海农业科技,1991, (2): 56
[119]郑平,张雄坚,陈应东等.广东甘薯组培脱毒苗田间比较试验初报[J].广东农业科学,1991,5:12-14.
[120]周桂珍.京郊大蒜病毒病原的研究及其鳞茎中病毒的脱除[J].植物病理学报,1989,10(3):145-148
[121]周桂珍,曹鸣庆,裘季燕等.京郊大蒜病毒的研究及其鳞茎中病毒的脱除.[J]植物病理学报,1989,19(3):145-149
[122]中国农业科学院蔬菜研究所.中国蔬菜栽培学[M].北京:农业出版社,1987:319-326.
[2] Amasino RM. Vernalization and flowering time [J].Curr Opin Biotech, 2005,16: 154-158
[3] Ayabe M,Sumi S.Establishment of novel tissue culture method,stem-disc culture, and its practical application to micropropagation of garlic(Allium sativum L.)[J].Plant Cell Reports,1998,17:773-779
[4] Baker R,etal.ln:kruse PF Jr,Patterson M K Jr.eds. Tissue Culture: Methure: Methods and Application[J].New York:Academie Press,1973,735-739
[5] Ball E M and Barke M K.leaf-dip serology for electron microscope identification of plant viruses[J].Virology,1968,36:152-155
[6] Burdon R H.Superoxide and hydrogen peroxide in relation to mammalian cell proliferation[J].Free Radical Biology and Medicine,1995,18:775-779
[7] Bertaeeini A,Botti S,Tabanelli D,et al.Micropropagation and establishment of mite borne virus free garlic(Allium sativum L.)[J].Acta Horticulturae,2004,6(31):201-206
[8] Camara ,M Pasqual, JEBP Pinto. Obtaining plants from garlic(Allium sativum L.)cultivars Chinese and Chonan using in vitro cultured meristems[J].Cienciae Pratica. 1989,13(1):97~101
[9] Chen J,Zheng H Y,Antoniw J F,Adams M J,Chen J P,Lin L.Detection and classification of allexiviruses from garlic in China [J].Archives of Virotogy,2004,149,435-445
[10] Choi S L,Paek K Y,Kwun K C.Effect of explants source and shoot trimming on shoot multiplication and bulb formation of garlic in vitro[J].Jounal of the Korean Society for Horticultural Science,1985,26(4):304-312
[11] Conci V ,Nome S F,Milne R G.Filamentous viruses of garlic in argentina [J] . Plant Dis,1992, 76:594-596
[12] Dare K C,Oberley T D,Mouse K E,et al . Expression of manganese superoxide dismutase Promote cellular differentiation[J].Free Radical Biology and Medicine,1994,16:275
[13] Derrick K S.Qauntitative assay for plant viruses using serologically specific electron microscopy [J].Virology,1973,56: 652
[14] Du Y Q, Zhu J ZH,Shen W P.Study on virus elimination technology of garlic in Jiading by shoot tip culture[J].Acta Agriculturae Shanghai,2004,20(l):9-12
[15] Haque M S,Wada T,Hattorik.Garlic roots for micropropagation through in vitro bulblet formation[J]. Aeta Horticulturae,2000,No.520,45-52
[16] Hatta T,Franclz R L B.Cytopathic structures as-sociated with tonoplasts of plant cells infected with cucumber mosaic and tomato aspermy virus[J].Journal of General Virology ,1981,53: 343-347
[17] Hernandez PR,Prado O,Gil DV.1994.Production of virus-free garlic(Allium sativum L.)bymeristem culture[J].Centro Agricola.21(1):76~84
[18] Hus H T, Aebig J. Enzyme-linked immuunosorbent assay (ELISA)of plant viruses using protein A-enzyme conjugate and F(ab’)of immunglobin G(IgG)[J]. Phytopathology ,1982 ,74:708
[19] Kawarabayashi W,Asahira T.In vitro Multiplication of virus-free bublbs of Lilies [J].Japan Soc Hort Sci,1989,58:195-209.
[20] Khan N,Alam M S,Nath U K.In vitro regeneration of garlic through callus culture[J].Journal of Biological Sciences,2004,4(2):189-191
[21] Kim E K, Hahn E J,Murthy H N,et al.Enhanced shoot and bulblet proliferation of garlic(Allium sativum L.)in bioreactor systems[J].Journal of Horticultural Science and Biotechnology, 2004,79(5):818-822
[22] Kinard G R,Scott S W,Barnett O W. Detection of apple chlorotic leaf spot and apple stem grooving viruses using RT-PCR[J]. Plant Disease ,1996, 80(6): 616-621
[23] Komissarov V A.Bulblet culture of garlic.[J].Kartofel Ovoshchi,1997,(2):23-24
[24] Kudou R,Fujime Y,Amimoto K. Effects of plant growth regulators and sampling positions on organ formation of garlic.Technical Bulletin of the Faculty of Agriculture[J],Kagawa University,1995,47(l):15-22
[25] Lubraco G,Schubert A,Previati A. Micropropagation and mycorrhization of Allium sativum[J].Acta Horticulturae,2000,(530):339-343
[26] Ma Y,Wang H L,Zhang C J .High rate of virus free plantlet regeneration via garlic scrapes tip culture[J].Plant Cell Reports,1994,14(l):65-68
[27] Matsubars S, Chen D. In vitro production of garlic plants and field acclimatization [J]. Scientia Horticulturae,1989,4: 677-679
[28] MD Late Submission Abstracts in Vitro Biology Meeting[C].2005 meeting of the society for in vitro biology, June 5-7, Baltimore, 2005. 3028.
[29] Mohamed Y Y,Barringer S A, Splittstoesser W E.In vitro bulb production from Allium spp.[J].In Vitro Cellular and Developmental Biology Plant,1995,31(1):51-52
[30] Moriconi D N,Conci V C,Nome S F.In vitro plantlet regeneration from callus in garlic (Allium sativum L.)[J].Phytopathlogy Buenos Aires,1989,49(1-2):97-103
[31] Nagakubo T, Nagasawa A,Ohkawa H. Micropropagation of garlic through in vitro bulblet formation [J]. Plant Cell Tissue and Organ Culture,1993,32(2): 175-183
[32] Navarro L.Citrus shoot-tip grafting in vitro (STG) and its applications:a review Proc[J].Int. Soc. Citriculture,1981:l:452-456.
[33] Navarro L,Roistaeher N C,Murshige T.Improvement of shoot-tip grafting in vitro for virus-free citrus[J].Amer.Soc.Hort.Sci.,1975,100(5):471-479.
[34] Osaws K, Sugawara H. Studies on techniques of tissue culture一I .Experiment on the method of acclimatization of in vitro plantlets of vegetables [J]. Bulletin of Developmental Breeding Vegetables and Organic Crops,1980,7: 22-25
[35] Ploaie P G.Use of immune electron microscopy o detect viruses in plants regenerated from meristem tip cultures[J].Buletinul de Protectia Plantlor,1984
[36] Quak.F.,Applied and Fundamental Aspects of Plant Cell,Tissue and organ Culture.Ed.By J.Reinert andY.P.S.Bajaj,Springer-Verlag, Berlin,PP.1977:596-615
[37] Robledo P A, Villalobos A M , Jofre G E .Efficient plant regeneration of garlic (Allium sarivum L.)by root tip culture[J].InVitro Cellular and Developmental Biology Plant,2000,36(5):416-419
[38] Seabrook J A.In vitro propagation and bulb formation of garlic[J].Canadian Journal of Plant Science,1994,74(1):155-158
[39] Spiegel S,Scot S W,Bowman V,et al. Improved detection of Prunus necrotic ringspot virus by the polymerase chain reaction[J]. European Journal of Plant Pathology,1996,102(7) : 681-685
[40] Sumi S,Matsumi T,Tsuneyoshi T .Complete nucleotide sequences of garlic viruses A and C,members of the newly ratified genus Allexivirus[J].Archives of Virology,1999,144:1819-1826
[41] Suh S K,Park H G.Rapid multiplication through immature bulbil cultures of garlic[J].Journal of the Korean Society for Horticultural Sciencf,1993,34(3):173-178
[42] Takagi H, Qu Y.Effect of light quality, photoperiod and cold treatment on in vitro bulbing of garlic shoot tip [J]. Acta Horticulturae,1995,(393): 181-188
[43] Ucman R, Zel J, Ravnikar M. Thennotherapy in virus elimination from garlic: Influences on shoot multiplication from meristems and bulb formation in vitro[J]. Scientia Horticulturae,1998, 73(4):193-202
[44] Van Dijk P.Carlavirus isolates from cultivated Alliumspecies represent three viruses.Neth [J]Plant Pathol,1993,99: 233-257
[45] Van Dijk P.Survey and characterization of poty viruses and their strains of Allium species[J].Neth.J.Plant Pathil.,1993,99: 38-41
[46] Verbeek M,van Dijk P,van Well PMA.Efficieney of eradication of four viruses from garlie (Allium sativum L.) by meristem tip culture [J].European Journal of Plant Pathology,1995,101:3,231-239
[47] Walkey D.G.A, etal. 1987. Agronomic evaluation of virus-free and virus-infected garlic(Allium sativum L.)[J].J. Hort. sci.64(1):53-60
[48] Yasseen M Y, Walter E S, Richard E L. In vitro shoot proliferation of sets from garlic and shallot [J]. Plant Cell Tissue and Organ Culture,1994, 36: 243-247
[49]昌祖慧.大蒜病毒病及其防治方法[J]湖北植保,1995,(5): 20
[50]陈典.大蒜脱毒种苗培育技术研究[J].北方园艺,1997, (2): 29-30.
[51]陈炯,陈剑平.Allexivirus属的一个新成员—大蒜病毒E的基因组全序列测定和系统树分析.[J]科学通报,2001, (40):1463-1468
[52]陈建平.胶体金免疫电镜技术检测和鉴定病汁液中不同形态的植物病毒[J].植物生理学报,1993,23(2):169-174
[53]陈青奇,陈典,张海霞.大蒜育种研究现状[J].北方园艺,2006(2):40-41
[54]陈世儒,黄菊辉.大蒜离体快繁及脱毒[J].园艺学报,1991,3(3):133-137
[55]崔凯荣,邢更生,周功克.体细胞胚发生的生化基础[[J].生命科学,2001, 13(1): 28-33
[56]崔荣昌,等.大蒜花叶病原的鉴定和茎尖脱毒效果[J].中国蔬菜,1992,4:10-14
[57]崔月花.张彪.高红明.等.唐营蒲茎尖培养脱病毒的研究[J].江苏农业研究,2000,21(4):88-89.
[58]丁辛顺.脱毒大蒜苗的生产检测技术[J].上海农业学报,1998,4(1):23~28
[59]高山林,金雍安,蔡朝晖.大蒜分生组织培养脱病毒和快速繁殖技术[J].植物资源与环境学报,2000, 9(3): 5-18
[60]高述民,陆帼一.大蒜体细胞胚胎发生研究进展[J].植物学通报,2000,17(4): 338-344
[61]杭玲,潘颖南,黄卓忠.草墓甸旬茎尖组织培养与脱毒苗生产田[J].广西林业科学,1999,6:319-320
[62]洪健,李德葆,周雪平.植物病毒分类图谱(第一版)[M].北京:科学出版社,2001
[63]焦奎,张书圣.伏安酶联免疫分析法及其在植物血清学检测技术中的应用[J].化学通报,2000,(10): 50-55
[64]兰平,李文凤,朱水芳.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(自然科学版),2001,3:1-4.
[65]李亚男,陈大清.脱落酸和赤霉素对大蒜某些生理特性的影响[J].湖北农学院学报,16(2):93-97.
[66]李昌华,李小川,赵美华等.大蒜茎尖脱毒技术及组织培养研究[J].华北农学报. 1995,10(3):20-25
[67]李小川,赵美华.大蒜鳞芽生长点离体培养诱导小鳞茎的形成[J].山西农业科学,1996, 24(3):59-60
[68]刘华.环境条件对马铃薯病毒生物测定结果的影响[J].山西农业科学,2003,31(2): 72-74
[69]刘高琼,李式军,张学平.温光条件对离体大蒜鳞茎形成和内源激素变化的效应[J].园艺学报,1997,4(2):165-16
[70]陆关成,李方.余姚白蒜组织培养脱毒的研究.浙江农业学报,1991,3(3):133-137
[71]鲁宇文.侵染大蒜的线状植物病毒的外壳蛋白基因原核表达、抗血清制备及检测应用[M].浙江大学2006年硕士论文.
[72]栾雨时,奕非时,崔喜波.大蒜茎尖培养脱毒效果[J].北方园艺,1996(4):37
[73]栾非时,陈典,陈友.脱毒大蒜花原始体培养增殖技术的研究[J].中国蔬菜,1995. (3):4-6.
[74]刘华.环境条件对马铃薯病毒生物测定结果的影响[J].山西农业科学,2003,31(2): 72-74
[75]李向东,于晓庆,古勤生等.马铃薯Y病毒属病毒基因功能研究进展[J].山东科学,2006,(3):1-6
[76]马筠.植物病毒鉴定检测方法的研究进展[J].世界农业,2003,292: 50-51
[77]孟振农,陈永,赵启韬等.大蒜花序中不同繁殖器官的形态发生[J].山东大学学报(自然科学版),1997,32(3):332-336
[78]牛淑妍,焦奎,张成良.联苯胺-H202-HRP伏安酶联免疫分析体系测定植物病毒TMV和TRSV[J].分析科学学报,1998,17 (3):12-14
[79]彭宏梅,李月秋,张仲凯等.应用电镜技术研究大理州烟草苗期病毒病的发生情况.[J]云南农业大学学报,2000,15(1):71-72
[80]彭于发,王慧敏,彭有良.植物病理学研究[M].北京:中国农业出版社,1997.9-13.
[81]齐放军,贾敬芬,李继胜.抗氧化剂对首楷原生质体早期培养的影响[J].实验生物学报,1994,27(l):1-9
[82]佘建明,丁犁平,陆维忠等.大蒜体细胞诱导再生植株和小鳞茎及其移栽试验[J].江苏农业学报,1992,8(4):46-47
[83]施曼玲,周雪平.植物病毒的诊断技术[J].微生物学通报,2000,27(2): 149-151
[84]宋瑞琳,吴如健,柯冲.茎尖嫁接脱除柑桔主要病原的研究[J].植物病理学报,1999,29(3):275-279.
[85]宋元林,唐顺明等.大蒜、洋葱、葱、韭葱栽培新技术[M].北京:中国农业出版社. 1999.1~43
[86]土杰,张大伟,范卫红等.侵染西瓜的烟草花叶病毒(TMV)的生物学种类鉴定[J].安徽农业科学,2003, 31(6):972-974
[87]王文和,邓馨,胡文玉等.草幕叶片愈伤组织形成及再生芽分化的组织学研究[J].沈阳农业大学学报1999,30(4):430-433.
[88]王忠.植物生理学[M].北京:中国农业出版社,2000:415-417
[89]邢更妹,李杉,崔凯荣等.植物体细胞胚发生中某些机理探讨[J].自然科学进展,2000,10(8):684-692
[90]熊正琴,李式军,刘高琼等.大蒜花序轴离体培养的研究[J].南京农业大学学报,2000,23(3):25-28
[91]徐洁.指示植物鉴定马铃薯病毒技术的研究与应用[J].中国马铃薯,2002,(2) :73-75
[92]徐培文,孙慧生,孙瑞杰.脱毒大蒜速繁途径探讨和良繁体系的建立.中国蔬菜,1993,5:9-13
[93]徐培文,孙慧生,孙瑞杰等.大蒜茎尖培养脱毒及增产效果的研究[J].山东农业科学,1984,6:6-10
[94]徐培文,孙惠生,孙瑞杰等.大蒜脱毒技术及应用研究[J].中国农业科学,1998,31(2):92-94
[95]薛万新,陆帼一.大蒜蒜瓣离体繁殖研究[J].西北农业学报,1994, 3(3): 62-66
[96]徐品三,栗雨时,刘纪文等.百合不定芽培养脱毒种球生产的研究[J].植物学通报,2003,20(3):313-318.
[97]徐平东,李梅,林奇英等.应用A蛋白夹心酶联免疫吸附法鉴定黄瓜花叶病毒血清组[J].福建农业大学学报,1997,26 (1): 64-69
[98]徐平珍,刘涛,杨莹等.脱落酸在植物花发育过程中的作用[J].云南植物研究,2007,29(2),215-222.
[99]徐启江,陈典,张云修.分孽洋葱茎尖培养脱毒苗技术研究[J].西北农林科技大学学报,2002,30(2):102-106.
[100]薛光荣,杨振英,洪霓等.茎尖培养等处理脱除梨病毒的技术研究[J].中国果树,1996(3):9-11
[101]杨乃博.大蒜全展叶愈伤组织的诱导和植株再生[J].植物生理学通讯,1992(18):47-48
[102]杨有权:周清海:杨娟.大蒜瞒类害虫的研究[J]长江蔬菜1998,(3) :17-21
[103]杨有权,杨素真,王学国.大蒜病毒病及其防治[J]长江蔬菜.1997 ( 9) : 17-18
[104]于德才.大蒜茎尖脱毒及快繁研究[J].北方园艺,2005, (6):84-85
[105]远彤,李坷,闰文斌等.大蒜花芽分化的细胞组织学研究[J].河南农业大学学报,1996,30(2): 186-190
[106]运广荣主编.中国蔬菜实用新技术大全(北方蔬菜卷)[M].北京:科学技术出版社. 2004.515-518
[107]魏宁生,吴云峰.大蒜病毒病原的鉴定及组培脱毒研究[J].西北农业大学学报,1992,20(1): 76-81
[108]赵军良.植物茎尖培养与无毒种苗生产[J].北方园艺,1995,6:10-12.
[109]赵顺庆,李鹏飞,范怀忠.花叶病大蒜的茎端组织脱毒培养.华南农业大学学报,1987,8(4):l-8
[110]张昌伟,侯喜林,袁建玉等.太仓大蒜根尖离体培养直接诱导不定芽及其试管鳞茎的形成[J].植物生理学通讯,2004(2):167-170
[111]张德玉,刘成林,尚化芳等.大蒜病毒病的发生及综合防治技术[J].蔬菜2006, ( 8):27
[112]张明厚,魏培文,朱俊华.大蒜茎尖组培苗的检测[J].东北农业大学学报,1999,30(2):105-110
[113]张素芝,李纪蓉.一种新型的大蒜快繁体系——花苞不定芽再生体系的建立[J].山东农业大学学报(自然科学版),2007,38(2):159-162
[114]张素芝,李纪蓉.影响大蒜气生鳞茎萌发的因素分析[J].四川农业大学学报,2006,24(2):148-151
[115]张素芝,李纪蓉.植物激素配比对大蒜茎盘愈伤组织再生体系的影响[J].种子,2006(6):38-40.
[116]张明厚,魏培文,朱俊华等.大蒜茎尖组培苗的检测[J]东北农业大学学报,1999, 30(2), 105-110
[117]张仲凯,李毅.云南植物病毒[M].北京:科学出版社,2001
[118]郑海柔.大蒜试管苗中鳞茎的诱导[J].上海农业科技,1991, (2): 56
[119]郑平,张雄坚,陈应东等.广东甘薯组培脱毒苗田间比较试验初报[J].广东农业科学,1991,5:12-14.
[120]周桂珍.京郊大蒜病毒病原的研究及其鳞茎中病毒的脱除[J].植物病理学报,1989,10(3):145-148
[121]周桂珍,曹鸣庆,裘季燕等.京郊大蒜病毒的研究及其鳞茎中病毒的脱除.[J]植物病理学报,1989,19(3):145-149
[122]中国农业科学院蔬菜研究所.中国蔬菜栽培学[M].北京:农业出版社,1987:319-326.