Agrobacterium-mediated co-transformation of chestnutsomatic embryos with genes encoding for a chitinase and a thaumatin-like protein

Castanea sativa Mill.(sweet chestnut, European chestnut) is a tree species with a wide distribution and an important economic role in Europe, covering an area of over 2 million hectares (Conedera et al. 2004). The ink disease (caused by Phytophthoraspp.) and chestnut blight (caused by Cryphonectriaparasitica) are the two most important diseases that affect European chestnut. Genetic transformation of the disease-prone Castanea species with antifungal or antimicrobial genes to increase disease resistance or tolerance could be the initial step towards a reliable and complementary biotechnological alternative to conventional breeding efforts. The most widely used approach is the overexpression of pathogenesis-related (PR) proteins such as chitinases and thaumatin-like proteins. PR proteins are de¿ned as proteins encoded by the host plant induced under pathological or related conditions (Veluthakkal and Dasgupta 2010). In the present work the objective was to obtain chestnut somatic embryos and plants that overexpress chestnut chitinase (CsCh3) and thaumatin-like protein (CsTL1) genes. Explants consist of small clumps of two to three somatic embryos in globular or early-torpedo stages were co-cultured for 5 days with Agrobacterium tumefaciens strain EHA105 harbouring with pK7WG2D-CsCh3 or pK7WG2D-CsTL1 binary vectors. Each plasmid also contains the neomycin phosphotransferase (nptII) selective gene and the green fluorescent protein (egfp) reporter gene. Vectors were introduced to EHA105 separately so each Agrobacteriumclonehad only one vector. Co-transformation was performed by mixing equal concentrations of two Agrobacteriumclones transformed with each vector. The fluorescent protein simplified and improved the evaluation of transformation events as compared with the GUS assay used in previous protocols in chestnut transformation. The transformation efficiency, determined in basis to the fluorescence of surviving explants, was clearly genotype-dependent and the rates w