Agrobacterium-mediated transformation of European chestnut somatic embryos with a Castanea sativa (Mill.) endochitinase gene

Abstract

Chestnut blight, caused by Cryphonectria parasitica, is a severe disease that has devastated chestnut stands in North America and Europe. Genes encoding hydrolytic enzymes such as chitinases, which can degrade fungal cell wall components, are attractive candidates for improving disease resistance. This report describes a reliable and efficient protocol for the Agrobacterium-mediated transformation of somatic embryos of European chestnut with the endogenous CsCh3 gene that codes for chitinase. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was genotype-dependent. Although somatic embryos of all three lines evaluated were transformed, the best results were obtained with somatic embryos derived from line CI-9 (20%). The addition of silver thiosulphate (20 or 40 μM) improved the transformation efficiency of somatic embryos derived from lines CI-3 and CI-9, although the differences were not significant. A total of 88 independent transformed lines were obtained. The presence of transgenes was confirmed by green fluorescent protein (GFP) expression, PCR and Southern blot analysis. Transgenic lines were maintained by secondary embryogenesis or cryopreservation following vitrification procedures. Maturation and germination of transformed somatic embryos yielded transgenic plants. Fluorescence indicating overexpression of the transgenes was observed in somatic embryos and also in shoots and leaves. No phenotypic differences were found relative to control plants, suggesting a lack of any cytotoxic effects of the GFP.