Cryopreservation Of F.pentapylla. And Virus-infected F.ananassa And Genetic Stability Analysis

Cryopreservation is not only considered to be the most effective way to long-term preservation ofwild plant germplasm resources,but also a highly efficient virus-free technology,“Cryotherapy”.In thispaper, we studied the procedure of Fragaria pentaphylla and “All-Star”strawberry shoot tips vitrificationcryopreservation, and “All-Star”strawberry materials were infected theStrawberry mild yellow edge virus（SMYEV）, we detected virus-free rate after Cryotherapy. Then, we detected the genome sequences DNAand analyzed the variation of DNA methylation level of samples between the treatment and the control.The main results below:1.The procedure of Fragaria pentaphylla shoot tips vitrification cryopreservation wasexplorato-ry studied. The results showed that: the shoot tips were cryoacclimatized at a low temperature （4℃） for3weeks; pre-cultured in the Pretreatment of solid culture medium (0.4mol.L^-1sucrose+7g.L^-1agar+MS, pH=5.8) after3days; treated with liquid pretreatment medium (0.4mol.L^-1sucrose+2mol.L^-1glycerin+MS, pH=5.8) for30mins at room temperature and treated in PVS₂(0.4mol.L^-1sucrose+15%（w/v） DMSO+15%（w/v） ethylene glycol+30%（w/v） glycerin+MS, pH=5.8)at0℃for60mins, thesurv-ival rate was highest up to79.7%. Regenerations differentiated normally.2. Distinction of the best vitrification cryopreservation procedure between “All-Star”strawberryand Fragaria pentaphylla were found that: the shoot tips of “All-Star”strawberry were cryoacclimatized at alow temperature （4℃） for4weeks; pre-cultured in the Pretreatment of solid culture mediumafter3days;treated with liquid pretreatment medium for30mins at room temperature and treated in PVS₂at0℃for60mins, the survival rate was highest up to64.3%, different plant material have different cryopreservationprocedure. Then, SMYEV weredetected by RT-PCR technology,wefound that virus-free rate was highestup to95%.3. The technique of amplified fragment length polymorphism （AFLP） was used to analyze thegenome sequences DNA of the treatments and controls, no variation of bands was found, which indicated that there were no changes in the genomic DNA sequences of Fragaria pentaphyllaand“All-Star”straw-berry after cryopreservation.4. The methyl-sensitive amplified polymorphism （MSAP） markers techlologe was used toanaly-sis the variation of DNA methylation level of samples between the treatment and the control. Theresults indicated that DNA methylation states of treatment materials reduced compared with controls, DNAmeth-ylation state ofFragariapentaphylla and “All-Star”strawberrywerereduced6.73%and3.83%.Methyla-tion and demethylation of Fragaria pentaphylla DNA were5.70%and12.43%,but, Methylationand dime-thylation of “All-Star”strawberry DNA were8.61%and12.44%.