摘要
【目的】培育‘阳光玫瑰’葡萄组培脱毒苗木,初步建立‘阳光玫瑰’葡萄组培脱毒快繁技术体系。【方法】采用MS为基本培养基,以植物生长调节剂6-BA、NAA和IBA为变量,接种后‘阳光玫瑰’葡萄组培苗的生长状况为因变量;通过热处理结合茎尖培养技术,在32℃预热处理7 d,再逐渐升温至37℃热处理30 d后,剥取茎尖进行培养,待获得完整植株时,利用RT-PCR检测方法对‘阳光玫瑰’葡萄组培苗进行病毒检测。【结果】‘阳光玫瑰’葡萄嫩茎段外植体经75%乙醇30 s+0.1%氯化汞8 min处理,外植体的污染率和褐化率最低;经消毒灭菌的外植体接种到添加含有6-BA和NAA的MS培养基上诱导萌发,在1.5 mg·L^(-1)6-BA和0.2 mg·L^(-1)NAA的培养基上萌芽率最高;将启动培养获得的无菌新芽,接种到含有6-BA和NAA的MS培养基中进行继代培养,在1.0 mg·L^(-1)6-BA+0.1 mg·L^(-1)NAA的培养基中单芽增殖效果最明显;把继代培养中生长健壮的单芽切下,转入添加IBA和NAA的1/2 MS培养基中进行生根培养,在添加0.4 mg·L^(-1)IBA和0.2 mg·L^(-1)NAA的1/2 MS培养基上生根效果最佳;采用热处理结合茎尖培养进行‘阳光玫瑰’葡萄组培苗的脱毒处理,热处理植株的成活率为78%,茎尖成活率为60%,经检测,再生植株不带葡萄卷叶病毒1(GLRaV-1)、葡萄卷叶病毒3(GLRaV-3)、葡萄病毒A(GVA)、葡萄斑点病毒(GFkV)、葡萄扇叶病毒(GFLV)。【结论】初步建立了‘阳光玫瑰’葡萄组培脱毒快繁技术体系,为‘阳光玫瑰’葡萄组培脱毒苗的工厂化生产提供了技术支撑。
【Objective】‘Shine Muscat’grape,with its unique advantages of seedless,high sugar content,rich rose fragrance and good flavor,surpasses the quality of other grape varieties and is welcomed by consumers.In recent years,‘Shine Muscat’grape has been widely introduced and planted in Zhejiang,Jiangsu,Anhui,Yunnan,Guangxi and other places,showing a broad market prospect.With the development of grape industry,the demand for‘Shine Muscat’grape seedlings is growing rapidly.However,in the cultivation of this variety in China,the plants generally show the symptoms of viral diseases,so that the yield and berry quality were seriously affected.The traditional way of grape seedling production to remove the virus is inefficient.Therefore,it is necessary to establish a rapid propagation technology system for virus-free seedlings of‘Shine Muscat’grape by combining tissue culture with heat treatment method.【Methods】Based on the heat treatment method,the virus-free zone of the stem tip can expand by inactivating the virus,and the stem tip has the advantage of taking small apical meristems to grow a whole plant.In this paper,the tissue culture seedlings of‘Shine Muscat’grape were treated by combining heat treatment with stem tip culture.Firstly,the stem segments with axillary bud area of new canes were used as explants,and they were disinfected by different disinfection methods.Then they were inoculated on MS medium containing different concentrations of plant growth regulators.After a few days,the pollution rate,survival rate,germination rate,subculture multiplication coefficient,rooting rate,root length and root number of explants were calculated.The best method of disinfection,disinfection time,start medium,subculture medium,rooting medium for explants was determined.Secondly,when robust tissue culture seedlings were obtained,they were heat-treated at different culture temperatures and durations.At the end of treatment,the stem tips were selected and cultured to grow into intact plants.Finally,the tube seedlings were sent to qualified institutions for virus detection by RTPCR detection.【Results】After disinfection with 75%alcohol for 30 s and 0.1%mercuric chloride for 8 min,the contamination rate and browning rate of stem explants were the lowest,and the survival rate was 86%;the explants were inoculated on MS medium containing 6-BA and NAA to induce germination,and the highest germination rate was on MS medium containing 6-BA 1.5 mg·L^(-1)and NAA 0.2 mg·L^(-1),which reached 86.4%;the sterile buds were inoculated on MS medium containing 6-BA and NAA.On the medium containing 1.0 mg·L^(-1)6-BA+0.1 mg·L^(-1)NAA,the effect of proliferation was the most obvious in the subculture,and the multiplication coefficient was 3.5;from subculture,the single buds with strong growth were separated and transferred onto the medium of 1/2MS with IBA and NAA for proliferation and rooting,and the effect of rooting was the best on the medium of 1/2MS with IBA 0.4 mg·L^(-1)and NAA 0.2 mg·L^(-1),the rooting rate was 86%,the average number of roots was 4.13,and the average root length was 3.89 cm.detoxification treatment of tissue culture seedlings by heat treatment was combined with shoot tip culture.After 18 days of heat treatment at 37℃,individual plants began to gradually lose their green leaves and became withered,and finally the whole plant died.The survival rate of heat treated plants was 78%,and the shoot tip survival rate was 60%.The results showed that,regenerated plants did not contain grape leaf curl virus 1(GLRaV-1),grape leaf curl virus 3(GLRaV-3),grape virus A(GVA),grape spot virus(GFkV),and grape fan leaf virus(GFLV).【Conclusion】Through the technology of plant tissue culture,the production efficiency of grape seedlings could be improved effectively.At the same time,using heat treatment combined with detoxification technology of stem tip culture,the detoxification efficiency of virus in the tissue culture seedling was also greatly improved.Therefore,this study preliminarily established the technology system of tissue culture and detoxification and rapid reproduction of‘Shine Muscat’grape.Moreover,it can provide technical support for the factory production of detoxification tissue culture seedling of‘Shine Muscat’grape.It can also provide the market with high quality,robust,virus-free,excellent characteristics of virus-free‘Shine Muscat’tissue culture seedlings.
作者
林茜
高营营
覃换玲
黄天琨
赵宇
王钟霞
陈淑媛
LIN Qian;GAO Yingying;QIN Huanling;HUANG Tiankun;ZHAO Yu;WANG Zhongxia;CHEN Shuyuan(Biotechnology Reserach Institute,Guangxi Academy of Agricultural Sciences,Nanning 530007,Guangxi,China;Guangxi Plant Tissue Culture Seedling Co.LTD,Nanning 530007,Guangxi,China)
出处
《果树学报》
CAS
CSCD
北大核心
2021年第3期435-443,共9页
Journal of Fruit Science
关键词
‘阳光玫瑰’葡萄
组织培养
快繁
脱毒
‘Shine Muscat’grape
Tissue culture
Rapid propagation
Virus-free
作者简介
林茜,女,助理研究员,主要从事香蕉、葡萄、中草药等方面作物的组培及栽培技术的研究。Tel:0771-3243086,E-mail:657788823@qq.com;通信作者:高营营.Tel:18376788265,E-mail:595249583@qq.com。
