Droplet-vitrification for shoot tip cryopreservation of shallot (Allium cepa var. aggregatum): effects of PVS3 and PVS2 on shoot regrowth

Abstract

The present study described a droplet-vitrification cryopreservation for shoot tips of shallot (Allium cepa var. aggregatum), a small bulb onion. Shoot tips taken from in vitro stock shoots were precultured with 0.3 M and 0.5 M of sucrose, with 1 day for each concentration. Precultured shoot tips were treated with a loading solution containing 2 M glycerol and 0.6 M sucrose for 20 min and then exposed to plant vitrification solution 3 (PVS3) at 24 °C for 3 h of dehydration. Following exposure to PVS3, shoot tips were moved onto 5.0 µl PVS3 droplets on aluminum foil strips, followed by direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were thawed by incubation in liquid MS medium containing 1.2 m sucrose for 20 min at room temperature, and then post-thaw cultured for shoot regrowth. Exposure of the shoot tips to PVS3 produced shoot regrowth (58%). Differential scanning calorimetry (DSC) detected 1.8% of freezable water in the shoot tips that had been dehydrated by PVS2, and no freezable water in those by PVS3 treatment. Exposure to PVS3 provided a broader safe temperature range (− 196 °C to − 88 °C), compared to that (− 196 °C to − 116 °C) of PVS2, for cryopreserved samples. Histological observations found that PVS3 dehydration allowed many cells in the apical dome and in the leaf primordia to survive following freezing in LN, while PVS2 dehydration resulted in much fewer surviving cells in the apical dome. The droplet-vitrification cryopreservation produced 56%, 72% and 32% shoot regrowth in cryopreserved shoot tips taken from in vitro shoots, adventitious buds regenerated from stem discs and field-grown bulbs, respectively. Advantages and disadvantages of the use of different source explants for cryopreservation were discussed. The droplet-vitrification cryopreservation produced 45% and 70% shoot regrowth in the additional two shallot genotypes ‘Kverve’ and ‘Lunteviga’. The results obtained in this study provide technical supports for setting-up cryo-bankings of genetic resources of shallots and other Allium species. Establishment of a droplet-vitrification cryopreservation of shallot shoot tips provided technical supports for long-term preservation of diverse genetic resources and cryotherapy for virus eradication in shallot plants.