Cryopreservation of chestnut (Castanea sativa Mill.) zygotic embryos by encapsulation- vitrification

Conservation plays a fundamental role in the protection of plant species of ecological and economic importance. European chestnut (Castanea sativa Mill.) is one of the main and important species of forests in Georgia, especially in western Georgia. It is included in the Red List of Georgia and described as vulnerable species according to the IUCN category. Chestnut blight has been severe problem in Georgia for many years. One of the main reasons for chestnut disease is the fungus Cryphomectria (Endothia) parasitica, which causes chestnut cancer. In this regard, there is urgent need to develop effective ways for the conservation of C. sativa in order to preserve and restore chestnut forests. Cryopreservation in liquid nitrogen at -196 °C is the alternative method for the safe and cost-efficient long-term conservation of germplasm of hardwood species. This method has been broadly used for preservation of different biological material of hardwood forest species and hybrid clones. A new technique termed encapsulation-vitrification combines the advantages of vitrification and of encapsulation-dehydration and greatly reduces the time required for dehydration. So far, the encapsulation-vitrification technique was successfully applied to the cryopreservation of shoot tips of several fruit crops. The present study described for the first time a simple and efficient cryopreservation protocol for the cryopreservation of excised embryonic axes (EAs) of Castanea sativa L. by encapsulation–vitrification. Moreover, the application of an activated charcoal as a component of artificial matrix of synthetic seeds was tested with the aim to facilitate the optimization of an efficient cryo-procedure. The protocol described in this study can be successfully applied to a range of Castanea cultivars and hybrid clones for cryopreservation of Castanea germplasm.

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