红芽芋茎尖低温疗法脱毒的RT-PCR检测
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摘要
为了了解红芽芋茎尖低温疗法再生苗的脱毒情况,本研究对其进行芋花叶病毒(DsMV)、黄瓜花叶病毒(CMV)和芋羽状斑驳病毒(TFMoV)的RT-PCR检测。大田苗均检出芋花叶病毒(DsMV)和黄瓜花叶病毒(CMV);对于芋花叶病毒(DsMV),玻璃化法低温疗法脱毒率为50%,包埋玻璃化法低温疗法脱毒率为100%,包埋脱水法低温疗法脱毒率为50%,小滴玻璃化法低温疗法脱毒率为75%,茎尖常规培养脱毒率为75%,说明包埋玻璃化法低温疗法有利于脱除芋花叶病毒;对于黄瓜花叶病毒(CMV),玻璃化法低温疗法脱毒率和包埋玻璃化法低温疗法脱毒率均为100%,包埋脱水法低温疗法脱毒率为75%,小滴玻璃化法低温疗法脱毒率为75%,茎尖常规培养脱毒率为50%,说明4种低温疗法脱毒均有利于脱除黄瓜花叶病毒,但玻璃化法低温疗法和包埋玻璃化法低温疗法两种脱毒方式效果更佳。4种低温疗法再生苗、茎尖常规培养再生苗和大田苗中均未检出芋羽状斑驳病毒(TFMoV)。究其原因,可能采用的引物为甘薯羽状斑驳病毒引物,而甘薯和芋头是不同物种,可能病毒序列不一样,且NCBI基因库里没有FMo V的任何信息,完全没有参考序列设计引物,还有待于进一步研究。本实验可为红芽芋脱毒苗的规模化生产提供一定技术基础和理论依据。
In order to understand the detoxification of the plantlets regenerated from the shoot-tips of taro by cryotherapy,RT-PCR detections of its dasheen mosaic virus(DsMV),cucumber mosaic virus(CMV) and taro feathery mottle virus(TFMoV) were carried out in this paper.Dasheen mosaic virus(DsMV) and cucumber mosaic virus(CMV) were detected in field seedlings.For dasheen mosaic virus(DsMV),the detoxification rate of vitrification,encapsulation vitrification,encapsulation dehydration,droplet vitrification by cryotherapy,and shoot tip routine culture was 50%,100%,50%,75%,and 75%,respectively,indicating that the encapsulation vitrification by cryotherapy method was conducive to the removal of dasheen mosaic virus.For cucumber mosaic virus(CMV),the detoxification rate of vitrification by cryotherapy and encapsulation vitrification by cryotherapy,encapsulation dehydration by cryotherapy,droplet vitrification,and shoot tip routine culture was 100%,75%,75%,and 50%,respectively,which indicated that 4 kinds of cryotherapies were beneficial to the removal of cucumber mosaic virus(CMV),but the detoxification effect of vitrification by cryotherapy and encapsulation vitrification by cryotherapy was better.Taro feathery mottle virus(TFMoV) was not detected in 4 kinds of regenerated plantlets by cryotherapy,shoot tip conventional culture regenerated plantlets and field seedlings.The reason might be the primers used were sweet potato feathery mottle virus.Sweet potato and taro were different species,so their viral sequences were probably not the same.What's more,there were no information of FMo V in NCBI gene pool,and the primers were designed without reference to the sequence,so it remained to be studied further.The experimental results could provide technical and theoretical basis for large-scale production of detoxification plantlets of Jiangxi Yanshan red bud taro.
In order to understand the detoxification of the plantlets regenerated from the shoot-tips of taro by cryotherapy,RT-PCR detections of its dasheen mosaic virus(DsMV),cucumber mosaic virus(CMV) and taro feathery mottle virus(TFMoV) were carried out in this paper.Dasheen mosaic virus(DsMV) and cucumber mosaic virus(CMV) were detected in field seedlings.For dasheen mosaic virus(DsMV),the detoxification rate of vitrification,encapsulation vitrification,encapsulation dehydration,droplet vitrification by cryotherapy,and shoot tip routine culture was 50%,100%,50%,75%,and 75%,respectively,indicating that the encapsulation vitrification by cryotherapy method was conducive to the removal of dasheen mosaic virus.For cucumber mosaic virus(CMV),the detoxification rate of vitrification by cryotherapy and encapsulation vitrification by cryotherapy,encapsulation dehydration by cryotherapy,droplet vitrification,and shoot tip routine culture was 100%,75%,75%,and 50%,respectively,which indicated that 4 kinds of cryotherapies were beneficial to the removal of cucumber mosaic virus(CMV),but the detoxification effect of vitrification by cryotherapy and encapsulation vitrification by cryotherapy was better.Taro feathery mottle virus(TFMoV) was not detected in 4 kinds of regenerated plantlets by cryotherapy,shoot tip conventional culture regenerated plantlets and field seedlings.The reason might be the primers used were sweet potato feathery mottle virus.Sweet potato and taro were different species,so their viral sequences were probably not the same.What's more,there were no information of FMo V in NCBI gene pool,and the primers were designed without reference to the sequence,so it remained to be studied further.The experimental results could provide technical and theoretical basis for large-scale production of detoxification plantlets of Jiangxi Yanshan red bud taro.
引文
Brison M.,Pierronnet A.,an d Dosba F.,1997,Effect of cryopreservation on the sanitary state of a cv Prunus rootstock experimentally contaminated with Plum Pox Potyvirus,Plant Sci.,123(97):189-196
Chen F.,and Wang Z.C.,2006,New technique of plant virus-free-cryotherapy,Henan Nongye Kexue(Journal of Henan Agricultural Sciences),35(12):11-13(陈芳,王子成,2006,植物脱毒新技术---低温疗法,河南农业科学,35(12):11-13)
Chi H.R.,and Mao B.Z.,2017,Research advances on plant virus detection and virus-elimination methods,Shengwu Jishu T-ongbao(Biotechnology Bulletin),33(8):26-33(迟惠荣,毛碧增,2017,植物病毒检测及脱毒方法的研究进展,生物技术通报,33(8):26-33)
Hong S.R.,and Yin M.H.,2013,A simple cryopreservation protocol for in vitro-grown shoot tips of Chinese genuine red bud taro(Colocasia esculenta L.Schott Var.Cormosus CV.Hongyayu)by encapsulation-dehydration,Sci.Hortic.,162(3):226-233
Jiang Q.,Liao H.J.,Dong L.,Chen J.X.,Ning Z.Y.,Li W.W.,and Xu X.T.,2015,Induction of multiple shoot and establishment of regeneration system in Clocasia escalenta Schott,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(3):675-679(江芹,廖华俊,董玲,陈静娴,宁志怨,李卫文,许晓婷,2015,红芽芋的丛生芽诱导和再生体系建立,分子植物育种,13(3):675-679)
Liu W.H.,Chen J.S.,and Li Y.W.,2004,Molecular identification of araceae-infecting viruses and their ecology,Yingyong Shengtai Xuebao(Chinese Journal of Applied Ecology),15(4):566-570(刘文洪,陈集双,李永伟,2004,侵染天南星科植物病毒的分子鉴定及其生态学研究,应用生态学报,15(4):566-570)
Liu X.X.,and Cheng Z.H.,2013,Research progress on cryopreservation of garlic germplasm and its virus eradication technique,Zhongguo Shucai(China Vegetables),(2):12-19(刘晓雪,程智慧,2013,大蒜种质超低温保存及脱毒技术研究进展,中国蔬菜,(2):12-19)
Liu Y.P.,2008,The preliminary research on establishment of virus-elimination and rapid propagation of taro(Colocasia esculenta Schott.),Thesis for M.S.,Huazhong Agricultural University,Supervisor:Li H.X.,and Ke W.D.,pp.34-55(刘玉平,2008,芋脱毒快繁体系建立的初步研究,硕士学位论文,华中农业大学,导师:李汉霞,柯卫东,pp.34-55)
Sant R.,Panis B.,Taylor M.,and Tyagi A.,2008,Cryopreservation of shoot-tips by droplet vitrification applicable to all taro(Colocasia esculenta var.esculenta)accessions,Plant Cell Tiss.Org.,92(1):107-111
Sant R.,Taylor M.,and Tyagi A.,2006,Cryopreservation of in vitro-grown shoot-tips of tropical taro(Colocasia esculenta var.esculenta)by vitrification,Cryoletters,27(3):133-142
Shi S.M.,and Hong N.,2010,Research progress of taro virus disease,Changjiang Shucai(Journal of Changjiang Vegetables),(14):3-5(施世明,洪霓,2010,芋病毒病研究进展,长江蔬菜,(14):3-5)
Takagi H.,Thinh N.T.,Islam O.M.,Senboku T.,and Sakai A.,1997,Cryopreservation of in vitro-grown shoot tips of taro(Colocasia esculenta(L.)Schott)by vitrification.1.Investigation of basic conditions of the vitrification procedure,Plant Cell Rep.,16(9):594-599
Tao Y.,and Wu X.Q.,2017,Research progress on methods for the detection of plant virus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(7):2901-2906(陶源,吴兴泉,2017,植物病毒检测方法的研究进展,分子植物育种,15(7):2901-2906)
Wang A.P.,Yin M.H.,and Hong S.R.,2015,Cryopreservation of in vitro-grown shoot tips of red bud taro by encapsulation-vitrification,Zhiwu Fenlei Yu Ziyuan Xuebao(Plant Diversity and Resources),37(6):801-812(王艾平,尹明华,洪森荣,2015,红芽芋茎尖的包埋玻璃化法超低温保存,植物分类与资源学报,37(6):801-812)
Wang Z.C.,Qu X.,and Bo T.,2011,Elimination of two potato virus by cryopreservation,Henan Daxue Xuebao(Journal of Henan University),41(6):609-614(王子成,曲先,薄涛,2011,超低温保存脱除两种马铃薯病毒,河南大学学报,41(6):609-614)
Xu H.L.,and Liu H.Y.,2017,Research progress on cryopreservation of plant germplasm resources by encapsulation-dehydration,Guizhou Nongye Kexue(Guizhou Agricultural Sciences),45(5):119-122(徐海龙,刘华英,2017,植物种质资源包埋脱水法超低温保存研究进展,贵州农业科学,45(5):119-122)
Zeng L.,He M.J.,Wu Y.,Gu Y.K.,and Li R.T.,2017,Cryopreservation of 6 kinds of medicinal herbs seeds,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),38(6):1149-1154(曾琳,何明军,吴怡,顾雅坤,李榕涛,2017,6种草本药用植物种子超低温保存技术研究,热带作物学报,38(6):1149-1154)
Chen F.,and Wang Z.C.,2006,New technique of plant virus-free-cryotherapy,Henan Nongye Kexue(Journal of Henan Agricultural Sciences),35(12):11-13(陈芳,王子成,2006,植物脱毒新技术---低温疗法,河南农业科学,35(12):11-13)
Chi H.R.,and Mao B.Z.,2017,Research advances on plant virus detection and virus-elimination methods,Shengwu Jishu T-ongbao(Biotechnology Bulletin),33(8):26-33(迟惠荣,毛碧增,2017,植物病毒检测及脱毒方法的研究进展,生物技术通报,33(8):26-33)
Hong S.R.,and Yin M.H.,2013,A simple cryopreservation protocol for in vitro-grown shoot tips of Chinese genuine red bud taro(Colocasia esculenta L.Schott Var.Cormosus CV.Hongyayu)by encapsulation-dehydration,Sci.Hortic.,162(3):226-233
Jiang Q.,Liao H.J.,Dong L.,Chen J.X.,Ning Z.Y.,Li W.W.,and Xu X.T.,2015,Induction of multiple shoot and establishment of regeneration system in Clocasia escalenta Schott,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(3):675-679(江芹,廖华俊,董玲,陈静娴,宁志怨,李卫文,许晓婷,2015,红芽芋的丛生芽诱导和再生体系建立,分子植物育种,13(3):675-679)
Liu W.H.,Chen J.S.,and Li Y.W.,2004,Molecular identification of araceae-infecting viruses and their ecology,Yingyong Shengtai Xuebao(Chinese Journal of Applied Ecology),15(4):566-570(刘文洪,陈集双,李永伟,2004,侵染天南星科植物病毒的分子鉴定及其生态学研究,应用生态学报,15(4):566-570)
Liu X.X.,and Cheng Z.H.,2013,Research progress on cryopreservation of garlic germplasm and its virus eradication technique,Zhongguo Shucai(China Vegetables),(2):12-19(刘晓雪,程智慧,2013,大蒜种质超低温保存及脱毒技术研究进展,中国蔬菜,(2):12-19)
Liu Y.P.,2008,The preliminary research on establishment of virus-elimination and rapid propagation of taro(Colocasia esculenta Schott.),Thesis for M.S.,Huazhong Agricultural University,Supervisor:Li H.X.,and Ke W.D.,pp.34-55(刘玉平,2008,芋脱毒快繁体系建立的初步研究,硕士学位论文,华中农业大学,导师:李汉霞,柯卫东,pp.34-55)
Sant R.,Panis B.,Taylor M.,and Tyagi A.,2008,Cryopreservation of shoot-tips by droplet vitrification applicable to all taro(Colocasia esculenta var.esculenta)accessions,Plant Cell Tiss.Org.,92(1):107-111
Sant R.,Taylor M.,and Tyagi A.,2006,Cryopreservation of in vitro-grown shoot-tips of tropical taro(Colocasia esculenta var.esculenta)by vitrification,Cryoletters,27(3):133-142
Shi S.M.,and Hong N.,2010,Research progress of taro virus disease,Changjiang Shucai(Journal of Changjiang Vegetables),(14):3-5(施世明,洪霓,2010,芋病毒病研究进展,长江蔬菜,(14):3-5)
Takagi H.,Thinh N.T.,Islam O.M.,Senboku T.,and Sakai A.,1997,Cryopreservation of in vitro-grown shoot tips of taro(Colocasia esculenta(L.)Schott)by vitrification.1.Investigation of basic conditions of the vitrification procedure,Plant Cell Rep.,16(9):594-599
Tao Y.,and Wu X.Q.,2017,Research progress on methods for the detection of plant virus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(7):2901-2906(陶源,吴兴泉,2017,植物病毒检测方法的研究进展,分子植物育种,15(7):2901-2906)
Wang A.P.,Yin M.H.,and Hong S.R.,2015,Cryopreservation of in vitro-grown shoot tips of red bud taro by encapsulation-vitrification,Zhiwu Fenlei Yu Ziyuan Xuebao(Plant Diversity and Resources),37(6):801-812(王艾平,尹明华,洪森荣,2015,红芽芋茎尖的包埋玻璃化法超低温保存,植物分类与资源学报,37(6):801-812)
Wang Z.C.,Qu X.,and Bo T.,2011,Elimination of two potato virus by cryopreservation,Henan Daxue Xuebao(Journal of Henan University),41(6):609-614(王子成,曲先,薄涛,2011,超低温保存脱除两种马铃薯病毒,河南大学学报,41(6):609-614)
Xu H.L.,and Liu H.Y.,2017,Research progress on cryopreservation of plant germplasm resources by encapsulation-dehydration,Guizhou Nongye Kexue(Guizhou Agricultural Sciences),45(5):119-122(徐海龙,刘华英,2017,植物种质资源包埋脱水法超低温保存研究进展,贵州农业科学,45(5):119-122)
Zeng L.,He M.J.,Wu Y.,Gu Y.K.,and Li R.T.,2017,Cryopreservation of 6 kinds of medicinal herbs seeds,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),38(6):1149-1154(曾琳,何明军,吴怡,顾雅坤,李榕涛,2017,6种草本药用植物种子超低温保存技术研究,热带作物学报,38(6):1149-1154)