Efficiency of transformation of Polish cultivars of pea (Pisum sativum L.) with various regeneration capacity by using hypervirulent Agrobacterium tumefaciens strains.
Efficiency of transformation of Polish cultivars of pea (Pisum sativum L.) with various regeneration capacity by using hypervirulent Agrobacterium tumefaciens strains.
复制标题
使用高毒力根癌农杆菌菌株转化具有各种再生能力的波兰豌豆品种（Pisum sativum L.）的效率。
DOI：
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发表时间：
2005
影响因子：
2.4
通讯作者：
J. Kapusta
中科院分区：
文献类型：
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作者：
T. Pniewski;J. Kapusta
以几个食用和饲料豌豆品种为材料，建立了农杆菌介导的豌豆遗传转化方法，先前的特征是它们通过器官发生再生的潜力。最合适的外植体是未成熟胚的切片，包括胚轴和子叶的基部，这是容易受到农杆菌感染，并能够再生转基因植物。3株A.测试了根癌农杆菌：AgL 0、AgL 1和EHA 105。每一个都携带含有uid基因的二元载体pP 35 SGIB，其中内含子在35 S启动子的控制下，并且bar基因赋予对草丁膦的抗性。菌株AgL 0被认为是有效的大多数品种，其次是AgL 1和EHA 105。转化效率从0.7%到4.1%不等，这取决于品种和农杆菌菌株。特定豌豆品种的转化效率与其再生能力并不明显对应，再生能力尽管不可或缺，但并不是成功转化的关键参数。通过PCR检测豌豆基因组DNA中整合基因的存在。T-DNA被稳定地传递到后代，因为它是由Southern杂交证实。导入基因的活性通过组织化学GUS测定和通过涂叶或通过用除草剂Basta喷洒转基因植物来分析。
An Agrobacterium-mediated transformation method of pea has been developed for several edible and fodder cultivars of pea (Pisum sativum L.), characterized previously in their potential for regeneration via organogenesis. The most appropriate explant, which was susceptible to Agrobacterium infection and capable of regenerating transgenic plants, turned out to be a slice of an immature embryo, including the embryo axis and the basal part of a cotyledon. Three hypervirulent strains of A. tumefaciens were tested: AgL0, AgL1 and EHA105. Each carried the binary vector pP35SGIB containing the uid gene, with an intron under control of the 35S promoter, and the bar gene conferring resistance to phosphinotricin. Strain AgL0 was found to be efficient for the majority of cultivars, followed by AgL1 and EHA105. Transformation efficiency varied from 0.7 to 4.1%, depending on cultivar and Agrobacterium strain. The transformation efficiency of particular pea cultivars did not clearly correspond to their regeneration capacity, which--although indispensable--was not a critical parameter of successful transformation. The presence of integrated genes in pea genomic DNA was detected by the PCR. T-DNA was stably transmitted to the progeny, as it was confirmed by Southern hybridization. The activity of introduced genes was analysed by the histochemical GUS assay and by painting leaves or by spraying transgenic plants with the herbicide Basta.