羽衣甘蓝小孢子胚胎细胞结构变化及其植株再生  

Microspore embryogenesis cytological structures variation and plant regeneration of kale

作　　者：姚悦梅[1] 张振超[1] 山溪 肖燕[1] 朱建飞[1] 戴忠良[1] YAO Yue-mei;ZHANG Zhen-chao;SHAN Xi;XIAO Yan;ZHU Jian-fei;DAI Zhong-liang(Zhenjiang Institute of Agricultural sciences of the Ning-Zhen Hilly District,Jurong,Jiangsu 212400,China)

机构地区：[1]江苏丘陵地区镇江农业科学研究所

出　　处：《南方农业学报》2019年第5期924-931,共8页Journal of Southern Agriculture

基　　金：江苏省“333工程”科研项目(BRA2017148);江苏省农业科技自主创新项目[CX(18)3075]

摘　　要：[目的]研究组织培养过程中羽衣甘蓝小孢子胚胎细胞结构变化及其植株再生,为小孢子培养技术在羽衣甘蓝中的应用提供理论依据。[方法]以12个羽衣甘蓝品种为材料,采用LEICA倒置荧光显微镜研究小孢子热激后的胚胎细胞结构变化、胚胎发育过程及出胚率差异,运用透射显微镜观察胚性小孢子细胞核的融合过程,并分析不同培养基(B5分化培养基和MS分化培养基)及其琼脂浓度(0.8%、1.0%和1.2%)对胚状体成苗率的影响。采用遮盖方式驯化组培苗后移栽大田,统计其成活率。[结果]在12个羽衣甘蓝品种中,除Y4花蕾的小孢子未发育成胚状体外,其他品种花蕾的小孢子均发育成不同数目的胚状体。其中,Y1和Y2平均每个花蕾的出胚数较高,分别为11.84和10.36个;Y3、Y7和Y8平均每个花蕾的出胚数较少,分别为1.55、1.45和0.94个。对于出胚数多的品种,对称分裂是其小孢子细胞分裂的主要方式,小孢子发育形成子叶形胚状体的比例也较高;而出胚数少的品种易发生小孢子不对称分裂,最终形成较多的畸形胚,子叶形胚数量较少。32.5 ℃热激1 d即可启动小孢子细胞胚胎发育进程,经原胚、球形胚、心形胚、鱼雷形胚,最终形成子叶形胚。热激处理后培养2 d小孢子进行第一次对称分裂形成两个大小、形状相似的细胞;培养5~7 d后两个细胞(胚性小孢子)逐渐靠近并融合在一起,细胞核核膜紧靠在一起,随后聚结融合,核酸物质混合,融合早期形成类似花生形的细胞核结构。含不同浓度琼脂的B5分化培养基和MS分化培养基中胚状体成苗率排序均表现为0.8%琼脂<1.0%琼脂<1.2%琼脂,且同一琼脂浓度下,B5分化培养基的胚状体成苗率均较MS分化培养基的高。组培苗驯化后移栽大田,成活率可达100%。[结论]通过小孢子培养可快速有效获得羽衣甘蓝小孢子单、双倍体再生植株。在培养过程中,胚性小孢子细胞核融�【Objective】The aim was to study the cellular structure changes and plant regeneration during microspore culture of kale,and to lay a theoretical foundation for the application of microspore culture technology in kale.【Method】 Twelve kale varieties were used as experimental materials to study the changes of cell structure,embryonic development and embryonic rate after microspore heat shock by LEICA inverted fluorescence microscopy. The fusion process of two nuclei by transmission electron microscopy was observed. The effects of different differentiation media(B5 and MS)and agar concentrations(0.8%,1.0% and 1.2%)on embryoid seedling formation in culture medium were also studied. The seedlings were domesticated by covering method then transplanted into field and calculated the survival rate finally.【Result 】Among 12 kale varieties,the other varieties developed into different numbers of embryoids except genotype Y4. The average number of embryos per bud of Y1 and Y2 was higher(11.84 and 10.36)than others,but the genotypes of Y3,Y7 and Y8 developed fewer number of embryos per bud,which were 1.55,1.45 and 0.94 respectively. For easy embryogenic genotypes,symmetrical division was the main mode of microspore division and the proportion of cotyledon embryos was higher. But for the varieties developed into fewer embryos were prone to asymmetrical division,resulting in many abnormal embryos and fewer cotyledon embryos. After heat shock for 1 d at 35 ℃,microspores initiated the first cell division, and the embryogenic microspores formed proembryos firstly,then globular embryo,heart-like embryo,torpedo embryo, and developed into cotyledonary embryos finally. After 2 d of culture,the microspores underwent the first symmetrical division to form two cells of similar size and shape. The two nuclear membranes of 5-7 d two cells(embryogenic microspores) gradually approached,followed by coalescence and fusion in culture,then nucleic acids mixed together and a peanut- like structure formed. The order of the frequency of

关 键 词：羽衣甘蓝 小孢子培养 胚胎发育 细胞学观察 胚状体 核膜聚结 

分 类 号：S635.9[农业科学—蔬菜学]