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Title: Cryopreservation of ancient apple cultivars of Veneto: A comparison between PVS2-vitrification and dormant bud techniques

item Maurizio, Lambardi
item Benelli, Carla
item Previati, Alberto
item De Carlo, A
item Ozudogru, E
item Ellis, David

Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/1/2010
Publication Date: 2/1/2012
Citation: Maurizio, L., Benelli, C., Previati, A., De Carlo, A., Ozudogru, E.A., Ellis, D.D. 2012. Cryopreservation of ancient apple cultivars of Veneto: A comparison between PVS2-vitrification and dormant bud techniques. Acta Horticulturae. 908:191-198.

Interpretive Summary: One hundred years ago there were thousands of commercial apple cultivars in Italy. Now, only eight cultivars make up the large-scale apple production in Italy and 70% of the production is made up of only 3 cultivars. The complete loss of these ancient cultivars is occurring now. Veneto Agricoltura has found and established plants from over 180 ancient cultivars yet these ancient cultivars are maintained in field orchards which are at risk from diseases, pests, floods, droughts, wind, etc. The secure long-term preservation these ancient cultivars in a priority for the Italian people, the question is how best to provide this long-term preservation. We looked at two methods for the cryopreservation of these apple cultivars and compared the relative labor and time costs for each. While each method has advantages and disadvantages, the use of dormant bud material for the cryopreservation of the ancient apple cultivars would take almost 50% less time and labor.

Technical Abstract: When working with vegetatively-propagated fruit species, cryopreservation of in vitro shoot tips using PVS2-based vitrification procedures still remains the most commonly used approach. However, other techniques are available for specific applications, such as cryopreservation of dormant buds which can be chip budded onto plants in the greenhouse and rapidly transferred to the field. Our report describes a comparison between a PVS2-vitrification and the Malus dormant-bud procedure for the cryopreservation of ancient apple cultivars, recovered in Veneto (North-East of Italy) and preserved in the field by the Veneto Agricoltura regional agency. In the PVS2-vitrification method, after 3 weeks of cold-hardening (at 4°C) shoot tips were excised from the stock cultures and cold-hardened at 4°C for additional 2 days. The shoot tips were exposed to a pre-loading solution (2 M glycerol and 0.4 M sucrose) for 30 min at 25°C, treated with PVS2 for 60 or 90 min a 0°C and then directly immersed in liquid nitrogen. After thawing, the shoot tips were plated on gelled medium. The combination of supplementing the recovery medium with1uM TDZ for 4 weeks, followed by hormone-free MS supplemented by activated charcoal for 3 weeks gave the best shoot tip regrowth. The best survival for 3 ancient cultivars (Campanin, Rosetta Mantovana and Pom dell’Oio) ranged from 57% to 100%. The dormant-bud protocol was based on winter collection of scions from which 35mm uni-nodal sections were cut, desiccated, slow cooled (-1°C/h) down to -30°C, stored in liquid nitrogen, thawed, re-hydrated and used for vegetative propagation by chip budding. Of three tested cultivars (San Piero, Canada Ruden and Rosa Gentil’), the best result was achieved with the cv San Piero, for which 79% of buds survived and 100% of the surviving buds regrew successfully when desiccated to 25% moisture content prior to cryopreservation and chip budding the buds onto clonal rootstocks. A cost-benefit analysis of the two crypreservation methods (PVS2-vitrification and dormant buds) revealed that the dormant bud method required 44% less time and labor.