Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system
Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system
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DOI：
10.1007/bf00232854
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发表时间：
1990-10
影响因子：
6.2
通讯作者：
N. Nehra;R. Chibbar;K. K. Kartha-K.;R. Datla;W. Crosby;C. Stushnoff
中科院分区：
文献类型：
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作者：
N. Nehra;R. Chibbar;K. K. Kartha-K.;R. Datla;W. Crosby;C. Stushnoff
建立了一种高效的草莓品种遗传转化方案。使用根癌农杆菌的红衣。该协议依赖于高频率(84%)的叶片再生系统。将非致癌的根癌农杆菌MP90菌株接种到叶盘上，该菌株携带双元载体pBI121，其中含有由诺帕林合成酶(NOS)启动子驱动的新霉素磷酸转移酶(NPT II)基因和由花椰菜花叶病毒35S(CaMV35S)启动子驱动的GUS标记基因。接种叶盘在非选择性不定芽再生培养基上预培养10d，在含50μg/ml卡那霉素的选择培养基上形成淡绿色分生组织区。在含有25μg/ml卡那霉素的第二选择培养基上，这些分生组织区域以6.5%的频率分化为转化芽。筛选出的不定芽在含卡那霉素的增殖培养基上增殖。所有试管苗均抗卡那霉素，表现出不同程度的NPT II和GUS酶活性。GUS活性的组织化学检测表明，35S启动子在茎和根尖的分生组织细胞中高度活性。对每个转基因克隆的分子分析证实，这两个标记基因都整合到了草莓基因组中。由转化植株制备的叶盘在卡那霉素上进行第二轮选择时，形成愈伤组织并表现出GUS活性。转化的生根植株被种植在温室中进行进一步的鉴定。该方法对草莓的基因改良具有一定的参考价值。
An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat usingAgrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenicAgrobacterium tumefadensstrain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 μg/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 μg/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.