Establishment Of Rapid Propagation System And Vitrification Cryopreservation System Of “XiangYe” Strawberry Shoot Tips

In this experiment,“Xiang Ye”strawberry stolon was used as test material,and tissue culture method was used to study the key links of explant sterilization,induction and differentiation culture,subculture and rooting culture,etc.The rapid propagation technology of“Xiang Ye”strawberry shoot tips tissue culture was established.The ultra-low temperature preservation system of"Xiangye"strawberry shoot tips by vitrification was optimized by orthogonal design and one-way ANOVA.The results are as follows:1.Establishment of tissue culture and rapid propagation system of“Xiang Ye”strawberry shoot tips:（1）Screening of disinfection methods for rapid propagation of strawberry shoot tips tissue culture:by comparing the different disinfection methods of strawberry stolon tip,the suitable disinfection methods for strawberry stolon tip were selected as follows:Strawberry stolons（2-4 cm at the top of the stolons）with strong growth and no pests were collected and soaked in beaker for 5 minutes with a small amount of detergent powder.The stolons were washed in tap water for 0.5 hours.Add 2-3 drops of Tween-80 to soak for 5 minutes and then rinse with tap water for 0.5 hours.Transfer the washed stolons to the super-clean worktable,wash them three or four times with sterile water,soak them with ethanol（75%）for 30 seconds,and wash them three or four times with sterile water.Sterilization with 10%sodium hypochlorite for 5 minutes and cleaning with sterile water for four or five times can effectively reduce the pollution rate and browning rate of shoot tips culture.The disinfection effect is the best,and the survival rate is 64.44%.（2）Screening of the best medium for rapid propagation of strawberry shoot tips tissue culture:by comparing the effects of different hormone proportions on tissue culture of shoot tips,we screened out:the medium of MS+TDZ 1.00mg/L+NAA 0.10mg/L+Sucrose 30g/L+Agar 7g/L,p H5.8 was the most suitable for inducing differentiation of strawberry shoot tips;the medium of MS+TDZ 0.75mg/L+NAA 0.2mg/L+Sucrose 30g/L+Agar 7g/L,p H5.8 was suitable for subculture of strawberry shoot tips,the concentration of TDZ should be gradually decreased with the increase of subculture times;the medium of MS+NAA 0.1mg/L+Sucrose30g/L+Agar 7g/L,p H5.8 was suitable for rooting of strawberry shoot tips.2.Establishment of cryopreservation system of“Xiang Ye”strawberry shoot tips by vitrification:（1）Screening of pre-culture methods in cryopreservation system of strawberry shoot tips by vitrification:by comparing the effects of different sucrose concentration and dark culture time on the survival rate of shoot tips after cryopreservation,the results showed that:strawberry shoot tips（0.5mm）were peeled off and inoculated on MS+7g/L agar medium,the survival rate of shoot tips was the highest when the sucrose concentration was 0.3 mol/L;dark culture was carried out at 4℃,the survival rate of shoot tips was the highest when dark culture time was set to 3 days.（2）Screening of pretreatment methods（loading and dehydration）for ultra-low temperature preservation of strawberry shoot tips by vitrification:by comparing the effects of different loading liquids and treatment time,vitrification liquor and treatment time on the survival rate of shoot tips,it was screened out that:LS loading solution（0.4mol/L Sucrose+2mol/L Glycerol+MS）was used to treat the shoot tips for 60 minutes at room temperature,and the survival rate of the shoot tips was the highest.At 0℃,PVS₃vitrification solution（50%（w/v）Glycerol+50%（w/v）Sucrose+MS）was used to dehydrate the shoot tips for 2hours,and the survival rate of the shoot tips was the highest.（3）Screening of recovery medium for ultra-low temperature preservation of strawberry shoot tips by vitrification:by comparing the effects of different restoring media on the survival rate and seedling rate of shoot tips after cryopreservation,the results showed that:MS+GA₃0.1mg/L+TDZ 1.0mg/L+NAA 0.1mg/L+Sucrose 30g/L+Agar 7g/L,p H at about 5.8.