小麦和拟斯卑尔脱山羊草(Aegilops speltoides Tausch.)异源融合体形成的技术体系建立  

Establishment of technical systems for the heterokaryon formation between Triticum aestivum L. and Aegilops speltoides Tausch.

作　　者：苏集华[1] 王莉[1,2] 秦金燕 姚占军[1] 

机构地区：[1]河北农业大学农学院/河北省种质资源重点实验室,河北保定071001 [2]石家庄以岭药业股份有限公司,河北石家庄050035

出　　处：《核农学报》2013年第11期1610-1616,共7页Journal of Nuclear Agricultural Sciences

基　　金：河北省应用基础研究计划重点基础研究项目(11960145D);河北省人力资源和社会保障厅博士后科研资助项目(2009);中巴政府间科技合作项目(16-408)

摘　　要：通过PEG诱导小麦和山羊草属间原生质体融合试验,建立高效的异源融合体形成技术体系,为小麦抗病育种提供中间材料。利用中性红、FDA、罗丹明B三种染色剂分别对小麦成熟胚及叶片和拟斯卑尔脱山羊草叶片的原生质体进行染色,通过观察比较不同染剂和材料的组合对原生质体的分辨效果,确定各个材料的最佳染剂,根据酶解时间对原生质体质量以及原生质体密度对融合率的影响,确定最佳酶解时间和融合密度。结果表明:FDA适于染色小麦成熟胚,紫外光下,有活力原生质体(不含叶绿体)发出绿色荧光;罗丹明B适于染色小麦和拟斯卑尔脱山羊草的叶片,紫外光下,原生质体发出红色荧光;中性红染色效果相对较差,故选用小麦成熟胚和拟斯卑尔脱山羊草叶片进行原生质体融合。原生质体解离时间以4h为宜,此时原生质体的活力高达86.2%;原生质体融合的最佳密度为10×106个·mL-1,此时异源融合率高达16.1%。利用筛选得到的FDA和罗丹明B分别对小麦成熟胚和拟斯卑尔脱山羊草叶片的原生质体进行染色,可有效辨别异源融合体;酶解时间4h、融合密度10×106个·mL-1时,可获得较高活力的原生质体,利于异源融合体的形成。Based on intergeneric protoplast fusion between Triticum aestivum L. and Aegilops L. induced by PEG, this experiment was intended to establish and improve the system for the heterokaryon formation, and provided intermediate materials for disease resistance breeding in wheat. In this study, the protoplasts from the mature embryo and leaves of Mingxian 169 （ Triticum aestivum L. ） and leaves of Y2155a （Ae. speltoides Tausch. ） were dyed with neutral red, FDA （fluorescein diacetate ） and rhodamine B, respectively. In this experiment, it was studied for the optimal dyes, the optimum enzymolysis time and fusion density by observing and analyzing related parameters about the resolving effects of different combination, livability and fusion rate of protoplasts. The results showed that wheat mature embryo protoplasts （excluding the chloroplasts） stained by FDA emitted green fluorescent light under UV light, otherwise protoplasts of Triticum aestivum and Ae. speltoides leaves glowed red fluorescent light by staining with rhodamine B, while neutral red was unsuitable for poor staining. So wheat mature embryo and Ae. speltoides leaves were selected for the protoplast fusion. The optimal enzymolysis time of protoplast isolation was 4h, and the livability of protoplast was up to 86.2%. The optimum density of protoplast fusion was 10 × 106celis· mL^-1, and the heteronuclear protoplast fusion rate reached up to 16.1%. It was concluded that FDA and rhodamine B were suitable for dying wheat mature embryo and Ae. speltoides leaves, respectively. Based on it, heterokaryons could be effectively distinguished. When protoplast isolation time was 4h and protoplast fusion density was 10 × 106cells · mL^-1 , high livability protoplasts were obtained and heterokaryons were formed more easily.

关 键 词：小麦 拟斯卑尔脱山羊草 原生质体 异源融合体 

分 类 号：S512.1[农业科学—作物学]