Cryopreservation Of Shoot Tips Of Strawberry（Fragaria×ananassa Duch） And Cryotherapy For Eradication Of SMYEV And SVBV

Establishment of an effective and convenient technique for strawberry germplasm conservation and virus elimination plays a positive role in the healthy development of strawberry industry in China.In the present study,droplet-vitrification and encapsulated-vitrification cryopreservation protocols were established for strawberry.The droplet-vitrification and encapsulated-vitrification protocols were successfully applied to eliminate strawberry mild yellow edge virus（SMYEV）and strawberry vein band virus（SVBV）.It provides new technical support for conservation of strawberry germplasm and production of virus-free seedlings.The main results are documented as followings:1.A droplet-vitrification cryopreservation for strawberry was established by optimizing pretreatment condition,sucrose concentration,time of exposure to PVS2,and size of shoot tip.Survival cells（46%）in cryopreserved shoot tips were observed by paraffin slices.Ploidy levels were analyzed by flow cytometry for the regenerated plants,and no ploidy variation was detected.The droplet-vitrification was successfully applied to 6 strawberry genotypes,with an average regrowth rate of 75.46%.The highest regrowth rate of 100% was ’Ning Yu’and the lowest regrowth rate of 38.13% was ’Tao Xun’.Compared with other reports,the regeneration rate was increased by 14.2 %.2.An encapsulation-vitrification cryopreservation for strawberry was established by optimizing pretreatment condition,sucrose concentration,time of exposure to PVS2,and size of shoot tip.Survival cells（80%）in cryopreserved shoot tips were observed by paraffin slices.Ploidy levels were analyzed by flow cytometry for the regenerated plants,and no ploidy variation was detected.The encapsulation-vitrification was successfully applied to 6strawberry genotypes,with an average regrowth rate of 79.16%.The highest regrowth rate of100% was ’Ning Yu’ Sheng Dan Hong’ and the lowest regrowth rate of 47.2% was ’Yan Li’.Compared with other reports,the regeneration rate was increased by 10%.3.SMYEV and SVBV were successfully eliminated by droplet-vitrification and encapsulated-vitrification protocols.’Ning Yu’ plantlets infected with SMYEV and SVBV were used.Shoot tips were excised and treated by droplet-vitrification and encapsulated vitrification protocols.Shoot tip culture was control.Regenerants were detected by RT-PCR.0.5-1.0 mm shoot tips had the highest virus-free rate,regardless of virus-free methods.For SMYEV and SVBV,the virus-free rate of shoot tip culture,drop vitrification and encapsulated vitrification were 60% and 66.67%,80% and 100%,66.67% and 80%,respectively.Comparied with shoot tip culture of strawberry,cryotherapy gave higher virus-free rate and avoided of excising tiny shoot tips.