Genetic transformation of Citrus sinensis (L) Osbeck for resistance to Candidatus Liberibacter spp

Author(s)

Eveline Carla da Rocha Tavano

• Document type: Doctoral Thesis
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Centro de Energia Nuclear na Agricultura (CENA/STB)
• Defense date: 2013-02-19

Examining board members

Adriana Pinheiro Martinelli; Helvecio Della Coletta Filho; Francisco de Assis Alves Mourão Filho

• Advisor: Beatriz Madalena Januzzi Mendes

Abstract

Huanglongbing (HLB) associated to Candidatus Liberibacter spp., which colonizes the phloem, is considered one of the most serious diseases of citrus. One important strategy to control this disease consists of producing transgenic plants expressing, in the bacteria colonization tissue, genes encoding antibacterial peptides. The objective of this work was to produce transgenic sweet orange plants expressing genes encoding the antibacterial peptide attacin A (attA) driven by phoem-specific promoters. The work started with the development of the gene constructs, containing the attacin A gene (with or without signal peptide) controlled by either sucrose transporter gene (AtSuc2) or phloem protein 2 gene promoters (AtPP2) from Arabidopsis thaliana, or phloem protein 2 gene promotor (CsPP2) from Citrus sinensis. The genetic transformation of C. sinensis \'Hamlin\', \'Valencia\', \'Pera\' and \'Natal\' cultivars was done via Agrobacterium tumefaciens. Epicotyls segments collected from in vitro germinated seedlings were used as explants. Transgenic plants were identified by PCR analyses. PCR positive plants were acclimatized and transferred to specific greenhouse. Integration and transcription of the attA gene was confirmed in acclimatized transgenic plants by Southern and Northern blot analysis, respectively. The attA gene expression was validated by RT-qPCR analysis. \'Hamlin\' transgenic cultivars containing the AtSuc2 or AtPP2 promoters controlling the expression of attA (with or without signal peptide) were propagated by grafting, for future evaluation of Candidatus Liberibacter asiaticus resistance (AU)

• FAPESP's process: 08/10426-7 - Genetic transformation of Citrus sinensis (L.) Osbeck for resistance to Candidatus Liberibacter ssp
• Grantee: Eveline Carla da Rocha Tavano
• Support Opportunities: Scholarships in Brazil - Doctorate