Development of the droplet-vitrification cryopreservation technique as a viable alternative to the dormant bud technique for recalcitrant pears

Abstract

The French INRA Pip Fruit Biological Resource Center is in charge of the maintenance, management, characterization and promotion of the traditional and scientific genetic resources of apple, pear, quince and related species. In 2010, the first cryopreservation trials were performed on apple and pear using the dormant bud technique with the main aim of cryopreserving a major part of the INRA Malus and Pyrus collections. However, some accessions, mainly from the Pyrus genus, sometimes responded badly to the dormant bud technique. To optimize the ability to cryopreserve the largest number of accessions in our collections, the droplet-vitrification technique was tested on two cultivars of Pyrus: ‘Williams’ and ‘Conference’, used as controls since 2010. Our objective was to evaluate this method under our experimental conditions and to compare it with the dormant bud technique. Experiments were carried out using samples from a batch of budsticks collected in January 2015, in order to test the impact of several critical steps in the process. With both techniques, the immersion in liquid nitrogen step appeared to have the most important impact on the final results, much more than dehydration and slow-freezing for dormant bud technique or LS and PVS2 steps for droplet-vitrification technique. Regenerated plants were obtained after droplet-vitrification. With some improvements, this method could be a viable alternative to the dormant bud technique and opens up the possibility of new development paths for the optimization of long-term preservation systems of our genetic resources collections.