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Genetic transformation efficiency of 'carrizo' citrange with two gene constructions

Genetic transformation is considered an important tool to be applied in citrus genetic improvement. However, transformation efficiency may vary according to several factors, including the genetic construction utilized. This work aimed to evaluate the transformation efficiency of 'Carrizo' citrange [Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck], containing two different gene constructions including the uidA (GUS) gene controlled by the promoters Arabidopsis thaliana phloem protein 2 (AtPhP2) and Arabidopsis thaliana sucrose transporter 2 (AtSuT2). Epicotil segments from in vitro germinated seedlings were used as explants. The nptII gene, which confers resistance to the antibiotic kanamycin, was used in the constructions as a selection agent to regenerate transgenic plants. X-GLUC histochemical analysis was performed in all regenerated shoots in order to verify the expression of the uidA gene. From the 4790 epicotyl segments utilized, 366 shoots had a positive reaction in the histochemical analysis, and were further grafted on in vitro grown rootstocks. Five out of these shoots, from each gene construction, were selected to perform PCR analysis, with specific primers for uidA gene sequence amplification. All selected plants evaluated by PCR confirmed gene integration. Transformation efficiency and number of non-transformed shoots, evaluated by histochemical analysis, varied among gene constructions utilized

biotechnology; Citrus spp.; uidA gene (GUS); genetic improvement; tissue culture


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