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首页> 外文会议>Proceedings of the 12th International Rapeseed Congress: Sustainable Development in Cruciferous Oilseed Crops Production >Transformation of microspore-derived embryos of winter oilseed rape (Brassica napus L.) using Agrobacterium tumefaciens
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Transformation of microspore-derived embryos of winter oilseed rape (Brassica napus L.) using Agrobacterium tumefaciens

机译：根癌农杆菌转化冬季油菜（Brassica napus L.）的小孢子胚

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Microspore-derived embryos (MDEs) of Brassica napus present suitable materials for introduction of foreign genes. MDEs generally exhibit a high regeneration potential. Also, as a result of chromosome duplication, the introduced trait can be evaluated in a homozygote. The aim of this study was to develop a method for delivery of foreign genes into winter oilseed rape microspore-derived embryos using Agrobacterium tumefaciens strains. 21-day-old MDEs of homozygous line DH O120 were conditioned for transformation by exposure to low temperature for 2 weeks. Next,they were put for a few days in the growth room and then inoculated with Agrobacterium. The A. tumefaciens strains EHA105 and LBA4404 were used for transformation, both carrying the binary vector pKGIB containing the GUS gene with an intron under control of CaMV 35S RNA promoter and the bar gene under control of NOS promoter as a selection marker. Plants regenerated from transformed MDEs were resistant to herbicide Basta as it was confirmed by a test of leaf painting. The PCR analyses confirmed the presence of bar gene in the genomic DNA of obtained plants. All plants of T0 generation were haploid before seed set. The chromosome doubling was conducted using the method of colchicine treatment of secondary axillary shoots.Plants of T1 generation obtained from seeds collected from primary transformants were analysed for presence of the introduced bar gene. The progeny plants of the transformant DH T-39 were all transgenic as they inherited the transgene from double haploid, homozygous parent. T-DNA was stably transmitted into progeny plants as it was confirmed by Southern-blot hybridization of revealing the integration ofT-DNA into winter oilseed rape genomic DNA.
机译：甘蓝型油菜的小孢子来源的胚胎（MDEs）提供了合适的材料来引入外源基因。 MDE通常表现出很高的再生潜力。同样，由于染色体复制，可以在纯合子中评估引入的性状。这项研究的目的是开发一种使用根癌农杆菌菌株将外源基因传递到冬季油料油菜小孢子来源的胚胎中的方法。通过暴露于低温2周使21天大的纯合系DH O120的MDEs适应转化。接下来，将它们在生长室中放置几天，然后接种农杆菌。使用根癌农杆菌菌株EHA105和LBA4404进行转化，它们都携带含有GUS基因的二元载体pKGIB，其中CaUS 35S RNA启动子控制内含子，而NOS启动子控制bar基因作为选择标记。通过叶画测试证实，从转化的MDE再生的植物对除草剂Basta具有抗性。 PCR分析证实了获得的植物的基因组DNA中bar基因的存在。 T0世代的所有植物在结实前均为单倍体。使用秋水仙碱处理次生腋芽的方法进行染色体加倍。分析从初级转化体收集的种子获得的T1代植物中是否存在导入的bar基因。转化体DH T-39的后代植物都是转基因的，因为它们从双单倍体纯合亲本继承了转基因。 T-DNA稳定地传递到后代植物中，这是通过Southern印迹杂交证实的，该揭示揭示了T-DNA整合到冬季油料油菜基因组DNA中。

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