Transformed somatic embryos of european chestnut to express cisgenic Castanea sativa (Mill.) chitinase gene using Agrobacterium tumefaciens

Trabajo presentado en el ANQUE ICCE BIOTEC, Science the key for a better life, celebrado en Madrid (España), del 1 al 4 de julio de 2014Chestnut blight, caused by the ascomycete fungus Cryphonectria parasitica (Murr.) Barr, is one of the major diseases of chestnut, affecting at both Castanea dentata (Marsh) Borkh (American chestnut) and C. sativa (MilI.) (European chestnut). At present, specific genes conferring resistance to blight disease are not knO\vn. An altemative to obtain resistant trees ,vould be the overproduction of pathogenesis-related (PR) proteins which play maj or role in natural defense against pests and pathogens. Among these, chitinases, belonging to the PR-3 family ofproteins, hydrolyze the p-l, 4 glycosidic bonds that link the N-acetylglucosarnine residues of chitin and playa direct role in plant defense by hydrolyzing chitin [1]. In European chestnut, an endochitinase of 32 kD (CsCh3), purified from mature cotyledons, showed in vitro antifungal properties \Vhen assayed against Trichoderma viride [2]. The aim of the present work is to achieve the overexpression of the native chitinase-like protein CsCh3 gene in embryogenic cultures of European chestnut via Agrobacterium-mediated transformation in order to obtain chestnut plants "ith increased potential resistance/tolerance to blight disease. For the transformation experiments, explants consist of small clumps of two to three somatic embryos in globular or early-torpedo stages form three chestnut embryogenic lines \Vere co-cultured for 5 days v.;th Agrobacterium tumefaciens strain EHAI05 harboring the binary plasmid pK7WG2D-Ch3. This plasmid also contains neomycin phosphotransferase 11 (nptlJ) gene as the selectable marker gene for kanamycin resistance and the green fluorescente protein gen (gfp) as reporter marker gene. The transformation efficiency, determined in basis on the fluorescence of surviving explants, ,vas clearly genotype-dependent ranging from 20% in CI-9 line to 4.2% in the C12-Hl line. A total of 175 independent transformed lines were obtained. The presence and integration of CsCh3 gene in genomic DNA \Vas determined by PCR and Southem blot analyses and CsCh3 gene expression \Vas evaluated by qPCR. Successful embryo proliferation \Vas achieved in all of the transgenic lines regenerated and plants \Vere recovered after maturation and germination of transformed somatic embryos. No phenotypic differences \Vere found ,,,ith relation to control plants, suggesting no potential cytotoxic effects of the GFP. The results reported in the present work on the oyerexpression of a native gene into chestnut somatic embryos could be considered as a first step toward the production of blight-disease tolerant chestnut plants. These plants should be denominated "cisgenic" because an intact native gene derived from European chestnut has been used.This research was partially funded by Xunta de Galicia (Spain) through the project 09MRU002400PR.Peer reviewe