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Citrus sinensis and citrus reticulata genetic transformation for resistance to Candidatus Liberibacter spp


Citrus is affected by several phytopathological problems that interfere with the crop productivity. The Huanglongbing (HLB) associated with Candidatus Liberibacter spp., which colonizes the phloem, was detected in Brazilian orchards and is causing several problems to the crop. The development of resistant cultivars through genetic transformation is been considered a valuable approach in order to control this disease. The Laboratório de Biotecnologia Vegetal, CENA/USP, has been working with citrus genetic transformation since 1999 and recently started a work to developed sweet orange and mandarins transgenic plants expressing genes which may influence HLB resistance. Gene constructs containing promoters which favors the gene expression in phloem tissue (AtSut, AtPhP2, and CsPhP2) driven antibacterial or antimicrobial genes such as attacin A, D4E1, and citrus defensin 1 are been tested. At the moment, we have sweet orange plants cvs. 'Hamlin', 'Valencia', and 'Pera' expressing attacin A and D4E1 under the control of AtSut or AtPhp2 promoters, acclimatized under greenhouse conditions. Recently, new gene constructs with the CsPhP2 promoter associated with attacin A gene and with the AtSut promoter associated with citrus defensin 1 gene were obtained. Therefore the objective of this project is to evaluate the transgenic plants which are acclimatized in greenhouse to resistance to HLB and obtain new transgenic plants of sweet orange and mandarins ('Fremont', 'Nules', and 'Thomas') cultivars with the available gene constructs. The genetic transformation will be performed via Agrobacterium tumefaciens, containing the plasmid pCAMBIA2201 with the expression cassette. Epicotyl segments collected from in vitro germinated plantlets will be used as explants in the genetic transformation experiments. The identification of transgenic adventitious shoots will be done by PCR. The PCR positive plants will be acclimatized and transfer to certified greenhouse for the culture of transgenic plants. Southern and northern blot analyses will be performed for confirmation of the gene integration and transcription. Transgenic lines will be inoculated with Candidatus Liberibacter asiaticus and analyzed via quantitative PCR for pathogen resistance evaluation. (AU)

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