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AN EFFICIENT AND INEXPENSIVE SALT SCREENING PROTOCOL FOR DEVELOPING MARKER FREE PHD45 TRANSGENIC RICE PLANTS DERIVED FROM MATURE SEED
AN EFFICIENT AND INEXPENSIVE SALT SCREENING PROTOCOL FOR DEVELOPING MARKER FREE PHD45 TRANSGENIC RICE PLANTS DERIVED FROM MATURE SEED
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机译:用于开发来自成熟种子的无标记PHD45转基因水稻植物的高效无盐筛选方案
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摘要
The present invention provides development of an improved protocol to screen the marker free transgenic rice plants during tissue culture if the phenotypic characteristics of the gene of interest are reported earlier. The protocol is validated by taking Pea DNA helicase 45 (PDH45) gene as example. The invention provides construction of a marker free binary vector and its successful application to produce marker-free transgenic rice plants. The invention also provides a screening method for selecting transgenic rice plants during tissue culture. To generate selectable marker-free transgenic rice plants, a reporter gene free binary vector pCAMBIA 1300 is used by cloning the gene of interest in place of hygromycin. Using this pCAMBIA 1300 binary vector with the PDH45 gene, marker-free T1 transgenic. rice plants expressing PDH are produced by Agrobacterium-mediated stable transformation using 200 mM NaC1 as a screening agent, during tissue culture. This salt screening protocol can be easily adopted and used to efficiently generate and screened marker-free transgenic rice plants which may improve the strategy. against. stress tolerance and public acceptance of GM rice.
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机译:如果目的基因的表型特征较早报道,本发明提供了改进的方案的开发,以在组织培养期间筛选无标记的转基因水稻植物。以豌豆DNA解旋酶45(PDH45)基因为例验证了该协议。本发明提供了无标记二元载体的构建及其在生产无标记转基因水稻植物中的成功应用。本发明还提供了用于在组织培养期间选择转基因水稻植物的筛选方法。为了产生无选择标记的转基因水稻植物,通过克隆目的基因代替潮霉素来使用无报告基因的二元载体pCAMBIA 1300。使用带有PDH45基因的无pT1转基因的pCAMBIA 1300二元载体。在组织培养过程中,使用200 mM NaCl作为筛选剂,通过土壤杆菌介导的稳定转化生产表达PDH的水稻植株。这种盐筛选方案可以很容易地采用并用于有效地产生和筛选无标记的转基因水稻植株,从而可以改善该策略。反对。转基因水稻的抗逆性和公众接受度。
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