Advances in somatic embryogenesis and genetic transformation of European chestnut: Development of transgenic resistance to ink and blight disease

Somatic embryogenesis (SE), which is considered the most efficient in vitro procedure for mass propagation of plants, shows great potential for use in forest tree improvement programs. This chapter presents a summary of recent advances made in the development of SE systems for European chestnut and hybrid chestnuts. As in most other woody species, immature zygotic embryos constitute the most suitable material for induction of SE in European chestnut. However, somatic embryogenesis has also been induced in leaf and shoot apex explants derived from axillary shoot cultures. Although the initial rate of induction of SE is low, a large number of somatic embryos can be obtained by secondary embryogenesis. An efficient protocol for the production of transgenic somatic embryos mediated by Agrobacterium co-culture with marker genes has been described for European chestnut. A number of parameters were evaluated with a view to maximizing the transformation efficiency. The transformation efficiency was not significantly affected by wounding, co-culture temperature or bacterial growth phase, but it was significantly influenced by other parameters such as strain/plasmid combination, co-cultivation time, selective agent, genotype and developmental stage of the somatic embryos. Genetic transformation experiments aimed at inducing tolerance to ink disease and blight disease have been performed with the thaumatin-like protein (CsTL1) and chitinase protein (CsCh3) genes, respectively. The presence of transgenes was confirmed by histochemical GUS assay, GFP, PCR and Southern blot analysis. The chestnut plants obtained are not transgenic sensu stricto, because the overexpressed genes are isolated in chestnut, and they could be considered cisgenic plants. Vitrification-based cryopreservation procedures have been successfully used with zygotic embryos and with untransformed and transformed somatic embryos.