12. From the field to the tank: The cryo-banking of ancient Italian apple germplasm by the dormant-bud technique

The cryopreservation of dormant vegetative buds, proposed for the first time by Sakai in early 1960s for winter-collected twigs of poplar and willow, was afterwards adapted by Forsline and co-workers to the conservation in liquid nitrogen of uni-nodal shoot segments of apple cultivars and used by the National Plant Germplasm System (NPGS), in the USA, to back-up in-field apple germplasm. In short, the dormant-bud protocol is based on winter collection of naturally cold-hardened scions from which 35 mm uni-nodal segments are cut, desiccated, slow cooled (−1 °C/h) down to −30 °C, and stored in liquid nitrogen. In late spring, thawed and rehydrated segments are transferred to the greenhouse in a cold box where the individual buds are removed from the segments and used for vegetative propagation by chip budding onto rootstocks. Cryopreservation by the dormant-bud technique has the advantage of allowing the direct transfer of accessions from the field to the tank and vice versa , i.e., with no passage in tissue culture, which is necessary with the PVS2-based vitrification technique before and after storage in liquid nitrogen. For this reason, the dormant-bud cryopreservation of apple takes about 40% of the time and 50% of the labour necessary with the shoot-tip vitrification approach. Recently, the technique has moved from the USA to Europe, where programs for the adaptation of protocols to different winter climatic conditions are in progress in Italy, Denmark, Germany and Switzerland. Italy, for instance, is the highest producer of apple in the EU, with a market of over 2,200,000 tons of fruits in the year 2011. However, only three cultivars (‘Golden Delicious’, ‘Gala’ and ‘Red Delicious’) contribute for 70% of this wide-scale commercial production. Hence, Italy, as well as other European countries, is facing loss of diversity in ancient apple fruit germplasm. Because of that, in 2000, Veneto Agricoltura, the agricultural agency of the Veneto region (Italy), started a program aimed at conserving ancient apple cultivars, before they were lost forever. This program is focused on the documentation of specimen trees from old cultivars and their propagation and maintenance in in-field collection. The clonal orchard contains today about 200 cultivars. In order to guarantee a safe duplication of this valuable germplasm, cryopreservation by the dormant-bud technique was recently included in the programme, in collaboration with the CNR-IVALSA Institute of Florence. Up to now, an effective cryopreservation protocol has been developed for 19 accessions, with up to 100% of plants regrown after they were chip-budded with buds recovered from the storage in liquid nitrogen. Additional cultivars are introduced yearly in experimentation, with a careful evaluation for each one of the time required to desiccate uni-nodal segments below 30% of moisture content. A specific experiment is currently in progress aimed at evaluating the possibility to bypass one or more of the several steps of the original procedure. Moreover, in order to speed up the development and optimization of cryo-protocols for all the accessions, two post-conservation pre-grafting tests (i.e., the TTC and the electrolyte leakage assays) are under evaluation, with the aim to investigate bud and cambium cell survival to liquid nitrogen, as well as the possible correlation between results from these tests and from plant regrowth after grafting with cryopreserved buds.