| Several strawberry (Fragaria ananassa.Duch) cultivars were chosen as materials to develop efficient transformation protocol. Some factors that affect the adventitious shoot regeneration from leaf disc in vitro and Agrobactium-meidiated transformation were examed. An efficient method of shoot renegeration and genetic transformation of strawberry has been developed. "Toyonoka", "Honeoye", "Dudla" and "Darselect" have been successfully transformed using a binary vector plasmid pBPCBFl which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven CBFl (CRT/DRE binding factor 1) gene, and transgenic plants were obtained. Results of molecular analysis by PCR and Southern blots as well as physiological tests of cold resistance, confirmed the integration of CBF1 gene into the genome of these cultivars.The main research results were as follows:I . Establishment of shoot regeneration system of strawberry cultivars.1. Genotype was the main factor influencing regeneration system of strawberry in vitro. 18 tested cultivars showed different regeneration capacities although all of them occurred adventitious shoots regeneration. "Toyonoka", "Honeoye", "Dudla" . "Xinxing No.2" , "6-40", "Xinxing No.l" , "Darselect", "Tongzi No.l", "Italy No.l" , "Linguosiji" and "Baojiaozaosheng" obtained shoot regeneration frequency of 100%~63.3%, while other cultivars as "6-42", "Xingdu No.2" and "Gosk" obtained 31.9%~4.3% frequency.2. Growth regulators play important roles in shoot inducing protocol. A. It was necessary for the regeneration to supplement cytokinin and auxin in the regeneration medium. B.Concentration of 6-BA (1.0, 2.0, 3.0, 4.0, 5.0 mg/1) influenced regeneration frequency significantly. Some genotypes required 6-BA of concentration lower than 3.0 mg/1 while others needed 4.0 or 5.0 mg/1 to reach a high regeneration frequency. C. 2,4-D was more effective than other three auxin-like substances when supplemented into medium with 6-BA, and low concentrations worked well. D. Compared with 6-BA, TDZ showed wide effective concentration range at about 1.0~8.0 mg/1. E. Both the concentration of TDZ and its combination with auxin-like substances affected shoot regeneration. F. It was considerate to add TDZ into medium to enhance the frequency of regeneration when genotypes with low regeneration activity were examed. G. Diffirent genotypes required different kinds of hormone and different concentratons.3. MS medium was the suitable basal medium for most of tested cultivars exeapt "Darselect", which preferred MS basal salts supplemented with vitamins of B5 medium; According to the same cultivar, leaf disc showed high regeneration frequency than petiole; Dark incubation was not necessary for shoot regeneration from explants. A dark period of 4~6 days enhanced the frequency of "Dudla" while did not affect the frequency of "Honeoye". There was not notable difference for the frequency of regeneration whether leaf discs were placed abaxial side up or down; The leaf disc regeneration of "Honeoye" and "Dudla" decreased when pre-cultured the seedlings grown in vitro in a low temperature of 4C. 4. On medium containing 20 mg/l kanamycin, the shoot regeneration capacity of "Honeoye" was inhibited. The growth of Agrobactium was inhibited completely with 375 mg/l carbennicilin supplemented in the medium, and the regeneration frequency began to decrease on this medium. 500 mg/l carbennicilin was toxic to leaf discs and reduced the shoot regeneration greatly. As a result, 20 mg/l of kanamycin and 400 mg/l of carbennicilin were established as the concentration effective for selection of shoots and inibition of Agrobactium growth.II. Agrobactium-meidiated transformation using CBF1 gene.1. Genotype was also the key factor influencing genetic transformation of strawberry. The transformation frequency of "Toyonoka", "Honeoye", "Dudla" and "Darselect" were 3.57%, 4.
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