Cryopreservation of apple buds to assure a backup copy for pre-base materials held in screenhouses

The “Centro Attività Vivaistiche” (CAV) in Faenza (northern Italy) preserves in screenhouses 20 different species, including pomaceous, stone fruits, kiwifruit, olive, grape, nuts, strawberry, berry fruits, artichoke and various rootstocks for a total of over 2,000 accessions and almost 5,000 potted plants. In order to limit conservation costs, it is often necessary to proceed with the elimination of obsolete cultivars from the certification system. Italy has implemented the new European directives regulating the nursery production which indicate that pre-base mother plants and pre-base materials can also be stored by cryopreservation. The use of this technique would therefore allow to reduce the costs of conservation of accessions by keeping a single plant in screenhouse from cultivars with poor marketing, but preserving in parallel some buds, in the form of microcuttings, in cryobank. For this reason, a collaboration was initiated between CAV and the CNR-IBE of Sesto Fiorentino (Firenze) aimed at exploring the possibility of applying the technique of cryopreservation of dormant buds to mutant clones from 5 apple polyclonal cultivars in conservation. The applied procedure consisted, in short, on the storage of scions for 2 months at -5°C; the preparation of 35-mm microcuttings; their dehydratation in a cell at -5°C, until reaching the values of 23% and 26% moisture content; the wrapping of groups of 5 microcuttings in aluminum foil, in turn wrapped in transparent film; the gradual cooling of -1°C every hour, until reaching -30°C, temperature at which they remained for additional 24 h; their storage in liquid nitrogen, inside cryoboxes; the warming to 4°C for 24 h, after which the microcuttings, freed from the wrappings, were stratified in moist peat and kept for 2 weeks at 2°C; the grafting, using the 'chip-budding' technique. The results of tests demonstrated the high effectiveness of the technique with 4 of the 5 cultivars, even when applied to microcuttings taken in winter from plants in screenhouse, i.e., in condition of low natural cold hardening.