The technical parameters were selected during the cryopreservation by droplet vitrification with axillary buds of Vaccinium"Misty"in vitro.The wide spectrum of this method was tested with to-tal 66 genotypes of Vaccinium including 8 spieces.The genetic stability of strains regenerated from 5 cultivars after cryopreservation was tested by ISSR with 10 primers.The results showed that the single axillary bud(length 1-1.5 mm)cut from 30-day-old shoots in vitro was cultured on subculture me-dium(WPM+0.3 mg/L ZT+7 g/L agar+30 g/L sucrose,SCM)and on preculture medium(WPM+7 g/L agar+0.3 mol/L sucrose,PCM)for 1 day,respectively,at(25±2)℃ in the dark.The axillary buds were treated in loading solution(WPM+2.0 mol/L glycerol+1.0 mol/L sucrose,LS)for 30 min(room temperature)and immersed into Plant Vitrification Solution Ⅱ(WPM+300 g/L glycerol+150 g/L ethylene glycol+150 g/L DMSO+0.4 mol/L sucrose,PVS2)for 40 min(0 ℃).The droplets(one bud per droplet)were formed on aluminum foil paper and quickly put into liquid nitrogen(LN).The material taken out from LN was treated immediately in unloading solution(WPM+1.2 mol/L su-crose,ULS)for 20 min(room temperature).The axillary buds were cutltured on SCM medium for regen-eration under the normal culture condition after 1 day in the dark(25±2)℃.The testing results of wide spectrum showed that the average survival rate and regeneration rate were 81.3％and 47％,respec-tively.The molecular marker detection results of 5 bilberry varieties showed no ploymorphic band.
维普
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