| Formosan sweetgum, mainly distributed in the area South of Qinling Mountain and Huaihe River, is one of the important native tree species in China. Due to its many desirable traits, such as fast growth, wide adaptability, high resistance and red leaves in autumn, formosan sweetgum is becoming popular in urban greenland as street tree in many towns and as ornamental trees in courtyard. However, the spiny fruits of the tree disintegrate very slowly and cause a nuisance on lawns and walks and sterility or fruitless breeding would greatly improve the usefulness of formosan sweetgum.By optimizing of Agrobacterium-mediated transformation system and genetic transformation, transgenic plantlets were finally obtained successfully with the stable integration of GUS gene in the genome of Liquidambar formosana Hance. Based on the improved systems, genetic transformation with PaFT genes was carried out, and the main results were as follows:1. The method of culturing bacteria was improved in the Agrobacterium-mediated transformation system of formosan sweetgum. We added 0μM,50μM, 100μM, 200μM AS in the AB suspensions, after comparison of the GUS transient expression and the induction rate of resistance buds, we discovered that the GUS transient expression was 100% in the AB suspensions added 100μM AS. After co-culture, sweetgum was cultured 30 days on the medium of 300mg/L Cef. and then selected on the selection medium of 25 mg/L Kan+300 mg/L Cef. The induction rate of resistance buds was up to 91.6±3.0% and the average number of sprouting plexus of each explants was up to 3.12±0.55.2. The effect of the time of pre-cuture on the transformation system of formosan sweetgum was compared. The sweetgum was pre-cutured respectively 1 d,3 d,5 d and 7 d. The GUS transient expression and the induction rate of resistance buds were observed. As a result, the GUS transient expression was all 100% after pre-cutured 3d and 7d. After co-culture, sweetgum was cultured 30 days on the medium of 300mg/L Cef., and then selected on the selection medium of 25 mg/L Kan+300 mg/L Cef. The induction rate of resistance buds was 92.3±2.7% and 92.6±3.4%. And the average number of sprouting plexus of each explants was up to 3.23±0.01 and 2.98±0.56 respectively.3. The reducing the concentration of Kan in the selection medium and prolonging the time of selection was examed. The concentration of Kan was 0 mg/L,10 mg/L and 20 mg/L. and the time of selection was 0 d,10 d,20 d and 30 d was tested. As a result, sweetgum was cultured 30 days on the medium of 300mg/L Cef. and then selected on the selection medium of 25 mg/L Kan+300 mg/L Cef, the induction rate of resistance buds was 93.4±7.4%, and the average number of sprouting plexus each explants was 3.15±0.67,but the positive rate was very low. When the sweetgum explants were cultured on the selection medium with 10 mg/L Kan+300 mg/L Cef for 10 days, the positive rate was up to 88.9%.4. Based on the optimized Agrobacterium-mediated transformation system of formosan sweetgum, the transgenic plants were obtained.19 kanamycin resistant plants were positive in the PCR detection, and 2 transgenic plants expressed of GUS activity. It indicated that exogenous GUS gene was integrated into the genome of formosan transgenic plant..5. PaFT gene was used for transforming formosan sweetgum, PCR detection results show that 1 resistant plant was positive... |
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