Optimization of Droplet Vitrification Protocol for Cryopreservation of In Vitro Grown Blackberry Shoot Tips

Abstract

The droplet vitrification technique was adapted to blackberry 'Cacanska Bestrna' (Rubus fruticosus L.) by applying two different plant vitrification solutions as well as by optimizing the duration of vitrification treatments. Apical shoot tips were pretreated in liquid Murashige and Skoog (MS) medium with progressively increasing sucrose concentration (0.3 M for 15 h, then 0.7 M for 5 h), and subsequently loaded in solution containing 1.9 M glycerol and 0.5 M sucrose for 30 min. After loading, explants were dehydrated for 20, 30 and 40 min on ice with vitrification solution A3 (glycerol 37.5%, dimethyl sulfoxide 15%, ethylene glycol 15% and sucrose 22.5%) and for 40, 50 and 60 min at room temperature with PVS3 solution (glycerol 50% and sucrose 50%). Explants were frozen in individual microdroplets of vitrification solution by direct immersion in liquid nitrogen. After rapid rewarming performed by direct immersion in unloading solution containing 0.8 M sucrose for 30 min, explants were placed onto solid MS medium supplemented with 1 mg/L N-6-benzyladenine and 0.1 mg/L indole-3-butyric acid, cultivated in the dark for 7 days and than transferred to standard growth conditions. Under the described experimental conditions, survival and regrowth of cryopreserved shoot tips dehydrated on ice with A3 solution ranged between 45.0-90.9 and 25.0-61.8%, respectively. Longer exposure of explants to A3 solution brought about higher survival and regrowth rates. On the other hand, dehydration with PVS3 resulted in considerably higher survival rates (90.0-95.9%) and higher regrowth rates (77.3-90.0%) of cryopreserved explants. The shortest PVS3 treatment gave the highest survival and regrowth. The results obtained prove the feasibility of PVS3-based droplet vitrification technique for long-term storage of this genotype, while further research will focus on evaluation of the optimized protocol for its applicability to different genotypes of the Rubus genus.