| Strawberry(Fragaria ananassa Duch), perennial herbaceous plant in Rosaceae family, which is one of the major fruit crops and widely grown in the world. However, safe hibernation and deficiency of cold tolerance varieties are outstanding problems in strawberry production. Many strawberry cultivars exhibit octoploid and highly heterozygous, which causes difficulties to breed new varieties with the conventional cross-breeding. Modern genetic engineering improves some specific traits directly in the DNA level for breaking the boundaries between species, which play an increasingly role in producing strawberry varieties with disease resistance, insect resistance and herbicide resistance. RdreB1BI gene is important transcription factor which related to plant development and physiology. It is investigated that RdreB1BI transcription factors are able to improve tolerance to drought, high-salt and cold stresses in Arabidopsis. RdreB1BI was used as target gene, the transformation of strawberry by Agrobacterium-mediated was performed in this study. The results were as follows:1.Taking the leaves of strawberry cultivars 'Meiho' and 'Benihope' as experimental material, we have compared the effects of different basic media, dark culture time, hormone concentration, leaf age and way of placement on inducing adventitious shoot of strawberry. The results showed that regeneration of adventitious shoots of different cultivars have different optimum conditions. The best medium of 'Meiho'was MS+TDZ(2.5 mg/L)+IBA(0.1 mg/L)+2,4-D(0.1 mg/L), the optimum leafages were 30～40 day, the regeneration rate was up to 82.8%; the best medium of 'Benihope' was MS+TDZ(2.0 mg/L)+IBA(0.1 mg/L)+2,4-D(0.1 mg/L) and the optimum leaf ages were 10-20 day, the regeneration rate was 80%. Dark culture for 14 days could improve the regeneration rate of adventitious buds. The leaves whose abaxial surface touched the medium were better than that of inaxial surface touched the medium. Adding AgNO3 of 8 mg/L and activated carbon of 1000 mg/L could promote the regeneration of adventitious buds2. Taking the leaves of strawberry cultivar "Benihope" as experimental material, we have optimized a series of factors such as hygromycin selection pressure, type and concentration of antibiotics, agrobacterium concentration, dip time and optimized co-culture time that affected genetic transformation system and obtained a higher efficient system. The results showed that the bacterial concentration was OD600=0.4; infection time was 6min; use 250mg/L of Cb as antibacterial antibiotics,5mg/L hygromycin as the selection of antibiotics and cultured for 3 days.3. To cultivate new germplasm of strawberry which tolerances to low temperature stress, we transformated a stress promoter rd29A inducing RdreB1BI gene into the'Benihope' strawberry. Five transgenic plants were examined with GUS, PCR and RT-PCR after transplanting and copy number of the target gene were detected using Inverse PCR methods. The results showed that the gene has been integrated in the genome of genetic plant and the copy number was from one to three.4. To test the resistance to cold stress of the five strains that had passed the GUS staining, PCR, RT-PCR and gene copy number detection, we put them in different temperature. The line with cold resistance can tolerate -4℃was selected and determined the index of malondialdehyde(MDA), proline, soluble protein and soluble sugar content, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity. The results showed that introduced RdreB1BI gene significantly reduced MDA accumulation in plants, increased of soluble protein and content of free proline, SOD and CAT activity, thus improved the cold tolerance in transgenic strawberry.The RdreB1BI gene have the highest expression at 0℃... |
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