| Blueberry are fruit-bearing perennial shrubs belonging to the genus Vaccinium, and fruits contain highamounts of antioxidants which are beneficial to health including Vitamin C, Vitamin E, Vitamin A, SOD,anthocyanins, protein and other particular elements. Anthocyanins in the fruits are able to improve eyesight,and blueberry fruits have the effect of Anti-aging, antioxidant, disease prevention, prevent of Urinary TractInfections and so on. Blueberry root is very short so that water stress effect serious. But traditional breedingapproaches for blueberry are time-consuming and labor-intensive due to its heterozygosity and polyploidywhich hamper improvement. Transformation has opened a powerful approach to introduce interest genesand accelerate the breeding process for many fruit crop. Blueberry is an especially suitable target forimprovement through direct gene transfer manipulations, since it has a polyploidy genome and is asexuallypropagated. Shoot in vitro regeneration from leaf of three blueberry genotypes 'Elliot', 'Northland' and'Fundy' were investigated, and shoot regeneration system of 'Northland' was optimized. BADH gene wastransformed into 'Northland' mediated with Agrobacterium. Confirmed by PCR, The plants with BADHwere obtained. The main results were as following:Three blueberry genotypes were preliminarily selected to fred the genotype with the highest regenerationfrequency for blueberry leaf. The results showed that leaf regeneration efficiency of blueberry cultivar'Northland' was apparently higher than the other two cultivars. Accordingly, with stronger regenerationability and better genetic stability, 'Northland' was selected for the next regeneration and transformationexperiment as experiment materials.The influence of the combination hormones to blueberry leaf regeneration was investigated. Theregeneration efficiency of 'Northland' leave from modified WPM supplemented with ZT 1.0mg/L was93.3%, following were CPPU, 95.3%. The last was BA, only 51%. When the concentration of NAAincreased, the leaf regeneration frequency of blueberry decreased. The other factors, method of inoculationand pH, also influence the leaf regeneration. When 'Northland' was up-placed in the medium of pH5.4, theregeneration frequency was much higher.Blueberry explant was sensitive to antibiotic so that high concentration of antibiotic in selectionmedium would inhibit shoot regeneration of blueberry, so in the experiment the 50mg/L Kan was used toinhibit the regeneration of untransformed buds. After co-cultivation, explant were washed three times inliquid regeneration medium with 500mg/L Cef and liquid regeneration medium without Cef, then blotteddry on sterile filter paper and placed on fresh selection medium with 300mg/L Cef which could inhibit Agrobactium growth.On basis of shoot regeneration system of 'Northland' leaves, several factors that influenced genetictransformation were studied. The result indicated that before inoculating Agrobactium explant waspre-cultured 2 days to contribute to genetic transformation. 3 days co-culture with Agrobactium tumefacienand co-culture medium supplyment with 100mmol/L AS were beneficial to enhance genetic transformationfrequency. When delayed 5 days to screen after co-culture, it could enhance transformation frequency.Leaves were dissected two times perpendicular to the midrib and not cutted off as far as possible. Because itcould reduce the damage to the leaves, genetic transformation was raised.Through the selection of Kan, 15 resistant plants were obtained. The two extrmities sequence of BADHgene was used as distinctive primer to test Kan-resistant plants by PCR assays, BADH genes wereintegrated into blueberry genomes. 1 plant of 7 Kan-resistant plants tested by PCR showed positive reaction.It was preliminery vertified that BADH genes had been successfully integrated into blueberry genomes.
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