In Vitro Culture Of Strawberry(Fragaria Ananassa Duch.) And Its Assessments Of Genetic Variation

To satisfy the increasing demand of intensive production,an efficienr micropropagation system,genetic stability evaluation,DNA methylation variation to the in virto plantlets and shoot regeneration from leaves of two strawberry（‘Akihime’and‘Benihoppe’）cultivars were carried out in this present study.The main results are as follow:1 Establishment and optimization of efficient propagationThe stolons of two varieties of strawberry were used as explants.At frist explants were cleaned through water,then them were sterilized 5⁶ min by 0.1%sublimate solutions and inoculated with MS+0.5 mg/L BA+0.2 mg/L IBA+30 g/L sucrose+7 g/L agar.MS medium supplemented with 0.5 mg/L BA+0.1 mg/L IBA+30 g/L sucrose+7 g/L agar gave the best propagation ratio for‘Akihime’and‘Benihoppe’strawberry,the proliferation coefficient of‘Akihime’was 4.9,the proliferation coefficient of‘Akihime’was 1.9 after 30 days.‘Benihoppe’strawberry seedlings were yielded on 1/2MS+0.2 mg/L IBA+30 g/L sucrose+7 g/L agar,rooting rates were 100%.‘Akihime’strawberry seedlings were yielded on 1/2MS+30 g/L sucrose+7 g/L agar,rooting rates were more than 90%.With acclimatization of 10 d in autoclaved soil,the plants showed87%of survival after transplantation.2 Genetic stability assessment of in vitro material and ISSR molecular makerDue to material limitation,the generation of 0¹2,3 were randomly selected for each generation,and 20 were randomly selected from 14^th generation and 18^th generation.ISSR markers were employed to evaluate the genetic stability at the 12^th cycle of transfer in‘Benihoppe’strawberry and 18^th cycle of transfer in‘Akihime’strawberry,respectively.The results showed that 207 bands were amplified from 49 primers of‘Benihoppe’strawberry,214 bands were amplified from 49primers of‘Akihime’strawberry.All the ISSR primers displayed the same profiles in vitro within 12cycles of‘Benihoppe’and 18 cycles of‘Akihime’.It is suggested that tissue culture can be used to expand the production in strawberry.3 DNA methylation variation assessment of in vitro material and MSAP molecular makerBy using 19 pairs selective-amplification primers of‘Benihoppe’strawberry and 21 pairs selective-amplification primers of‘Akihime’strawberry to investigate the in vitro shoots（5,7,9,10,11,12 subculture cycles and 1,2,3 months after trasfer）DNA methylation through MSAP molecular maker.2189 fragments were amplified by using 19 pairs of selective primers in‘Benihoppe’strawberry,2522 fragments were amplified by using 21 pairs of selective primers in‘Akihime’strawberry.In the 5^thh and 12^thh subcultures,the total methylation ratio of‘Benihoppe’strawberry was 11.3%to 9.3%and‘Akihime’strawberry was 10.5%to 8.3%.DNA methylation levels of in vitro shoots decreased along with the elongation of subculture cycles by MSAP.However DNA methylation levels in the asexual descendants of in vitro strawberry shoots increased gradually in the field.The effect of tissue culture on the DNA methylation levels was similar between the two strawberry cultivars.4 DNA methylation variation assessment of in vitro material were treated with low temperature and MSAP molecular maker2499 fragments were amplified by using 14 pairs of selective primers.The total methylation of DNA decreased across the cold treatment,total DNA methylation ratios were 27.2%,20.7%、25.8%,22.7%,22.7%and 22.4%in the samples exposed by cold stress for 0,1,3,5,7 and 10 days,respectively.However,the samples were recovered 5 days,total DNA methylation ratio increased26.05%.Furthermore,the demethylation was the main feature though both methylation and demethylation were triggerd by cold acclimation.Five methylated sites were extracted,cloned and sequenced finally.And NCBI Blast results showed that these fragments were homologous to functional genes which were the region of CCGG rich clusters.So these sequences demonstrated the results of DNA methylation by MSAP effectively.