Cryopreservation Of Blueberry (Vaccinium) Shoot Tips And Assessment Of Genetic Stability In Plants Regenerated From Cryopreservation

Blueberry(Vaccinium corymbosum)is one of the most commercially significant berry crops.Blueberry fruit,which are rich in anthocyanins and other bioactive substances,have received much attention due to their benefits to human health.Cultivated species and wild species are the prerequisites for traditional breeding and biotechnology breeding.However,due to serious environmental pollution,climate warming and other factors,the survival of plant germplasm resources is seriously threatened.Therefore,the preservation of plant germplasm resources are necessary.However,the traditional field preservation and test-tube preservation methods have their own limitations,and cryopreservation not only overcome the shortcomings,and easy to operate,insusceptible from pests and natural disasters and threats,can be long-term effective Preservation of plant germplasm resources.At present,the lack of genotype-wide-spectrum cryopreservation of blueberry germplasm resources system.We report a droplet-vitrification for efficient cryopreservation of valuable germplasm of blueberry.In this protocol,in vitro stock shoots were cold-hardened in the dark at 4oC for 3 weeks.Shoot tips(1.5-2.0 mm in length)including 4-5 leaf primordia were excised from the cold-hardened stock shoots and precultured with 0.3 M sucrose for 1 day.Precultured shoot tips were treated with a loading solution containing 1.0 M sucrose and 2 M glycerol for 30 min,followed by exposure to PVS2 for40 min at 0oC.Dehydrated shoot tips were then transferred onto 3.5?L PVS2 droplets on aluminum foil strips,prior to a direct immersion into liquid nitrogen for 1 h.Frozen foils with shoot tips were re-warmed in 1.2 M sucrose for 20 min at room temperature,followed by post-thaw culture for a recovery medium containing 0.3 mg L-1 zeatin for shoot regrowth.With optimized parameters,shoot regrowth levels were 46.7% for‘O'Neal' and 91.7% for ‘Misty',with a mean shoot regrowth level of 66.5 % obtained across the five valuable blueberry genotypes tested.Histological observations showed that many cells in the upper part of apical dome and some in leaf primordia 1-3 survived,while those in lower part of apical dome and leaf primordia 4-5 were seriously damaged,following cryopreservation.No polymorphic bands were detected using inter-simple sequence repeat markers(ISSR)andrandom amplified polymorphic DNA(RAPD)in regenerants of ‘O'Neal' and ‘Misty' recovered from cryopreservation.