A droplet-vitrification protocol enabled cryopreservation of doubled haploid explants of Malus x domestica Borkh. ‘Golden Delicious’

• First successful cryopreservation of apple doubled haploid apices by droplet-vitrification.
• Doubled haploids proved to be less tolerant to cryopreservation than the corresponding diploid variety.
• The use of meta-topolin reduced hyperhydricity of apple doubled haploid explants.

High throughput cryopreservation is a key step in the development of an apple t-DNA mutant library. The aim of this study was to develop an easy, efficient and rapid droplet vitrification protocol to cryopreserve doubled haploids of Malus x domestica Borkh. ‘Golden Delicious’. The doubled haploid genotype proved to be less tolerant to cryopreservation than ‘Golden Delicious’. Apices improved the quality of regeneration and the facility of dissection compared to meristems. About 50% of the explants treated with PVS2 for 40 min survived after immersion in liquid nitrogen, and 10% showed sustainable development. Phytotoxicity was observed mostly after immersion in the vitrification solution. Hyperhydricity was regularly observed in these experiments and decreased final efficiency. The use of meta-topolin instead of 6-benzylaminopurine (BAP) reduced hyperhydricity by almost 50%. We demonstrated that doubled haploid apple apices can be cryopreserved, but further improvements are needed before this technique can be used at high throughput.