High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration
High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration
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DOI：
10.1007/s00299-008-0646-2
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发表时间：
2009-03-01
影响因子：
6.2
通讯作者：
Otoni, Wagner Campos
中科院分区：
文献类型：
--
作者：
Peixoto de Oliveira, Maria Luiza;Febres, Vicente J.;Otoni, Wagner Campos
采用改良的农杆菌渗入法对菠萝甜橙子[Citrus sinensis（L.）Osbeck]和'Swingle' Citrumelo [Citrus paradisi Macf. X枳Poncirus trifoliata（L.）Raf.]是为了提高转化频率而开发的。比较了超声波辅助农杆菌介导转化法（SAAT）、真空渗透法以及两者结合法与传统农杆菌介导转化法（“浸渍”法）的转化效果。结果表明，上胚轴切段的形态发生潜力随着SAAT和真空处理时间的增加而降低。不同的SAAT处理对GUS瞬时表达没有影响，但真空浸润处理显著增强了GUS瞬时表达。当外植体经受SAAT 2 s和真空渗透10 min的组合时，获得最高的转化效率。PCR和Southern杂交分析uidA基因被用来证实转基因的整合。在这项研究中实现的转化频率（8.4%的'菠萝'甜橙子和11.2%的'Swingle'香柚）是最高的两个品种的报道。
An improved method for the Agrobacterium infiltration of epicotyl segments of 'Pineapple' sweet orange [Citrus sinensis (L.) Osbeck] and 'Swingle' citrumelo [Citrus paradisi Macf. X Poncirus trifoliata (L.) Raf.] was developed in order to increase transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum infiltration, and a combination of the two procedures were compared with conventional Agrobacterium-mediated inoculation method ('dipping' method). It was observed that the morphogenic potential of the epicotyl segments decreased as the duration of SAAT and vacuum treatments increased. Transient GUS expression was not affected by the different SAAT treatments, but it was significantly enhanced by the vacuum infiltration treatments. The highest transformation efficiencies were obtained when the explants were subjected to a combination of SAAT for 2 s followed by 10 min of vacuum infiltration. PCR and Southern blot analysis of the uidA gene were used to confirm the integration of the transgenes. The transformation frequencies achieved in this study (8.4% for 'Pineapple' sweet orange and 11.2% for 'Swingle' citrumelo) are the highest ones reported for both cultivars.