Establishment Of Efficient Regeneration System In Strawberry And Construction Of Project Agrobacterium Of Anti-herbicidal Bar Gene

The strawberry(Fragaria ananassa Duch)cultivars including Toyonoka,Kunouwase and shiyome suitable for cultivation in Anhui Province were taken as material to establish a simple and high-efficient regeneration system.Stem apex multiplication,callus induction from leaf,rooting culture and medium for transplant had been studied to provide a basic fundament for transgenic strawberry research.In order to supply project agrobacterium for bar gene transformation the hybridization of LBA4404 with HB101 and DH5a was done by triparental mating.Otherwise the system of bar gene transfection in strawberry was explored primarily.The main results gained were as follows.1.There was a significant difference in the different treatment of stem apex sterilization,of which the treatmentⅣwas best whit survival rate of 90%.2.In the culture of stem apex the survival rate was up to 95.6%when the size of explant was more than 0.7mm,lower than 20%when the size of explant was less than 0.3 mm,up to 80%and 84%respectively when the size of explant was 0.3～0.5 mm and 0.5～0.7 mm.3.The optimal multipling culture mediums for Toyonoka,Kunouwase and shiyome were MS+BA 0.5 mg/L+NAA 0.10 mg/L,MS+BA 1.0 mg/L+NAA 0.10mg/L,MS+BA 0.5 mg/L+NAA 0.20 mg/L respectively.It indicated that different genotype of strawberry had different optimal multipling culture.4.The best culture medium for the callus induced from leaf was MS+BA 3.0 mg/L+2,4-D 0.2 mg/L+IBA 0.3 mg/L by analysis of SPSS software.5.The optimal rooting culture mediums for Toyonoka,Kunouwase and shiyome were 1/2MS+IBA 0.1 mg/L,1/2MS+IBA 0.0 mg/L,1/2MS+IBA 0.2 mg/L.6.The result of transplant after plantlet training step by step showed that fermented rice husk and loam(1:1)was best transplanting medium resulting in the survival rate of 98%.And the other transplanting medium resulting in survival rates of 90%.7.Utilizing triparental mating hybridization of LBA4404 with HB101 and DH5a it was proved that the project agrobacterium with eukaryotic expression vector pBI121-bar had been constructed successfully by electrophoresis of products in PCR with bacterium colony and plasmid and in plasmid digested with XbaⅠand BamHⅠenzyme.8 The critical concentration of kanamycin for mortality of untransgenic strawberry plantlet and callus from leaf was 25 mg/L and 20 mg/L respectively9.The critical concentration Cef and Carb efficient for control of project agrobacterium growth were both at 200 mg/L.