| Apple (Malus domestica Borkh) is the one of the most important fruit trees in the world. Dwarf culture have became a general tendency of apple development, because which have some characters with early fruit, high yield, good quality and economic. Genetic transfer technology was employed to transfer ipt gene directed by different promtors into apple cultivars, which will have a high potency to get a new modification structure of apple trees reduced apical dominance, apted to dwarf culture and to accelerate the speed of apple breeding. It was significance of theory and reality that a high efficient transferring system was established and plant genetic engineering technology was applied to apple genetic improvement.In this study, high-frequency regeneration and transformation systems have been established through selecting the best conduction of regeneration, transformation and antibiotic selection of apple leaves in vitro, and ipt and SSU-ipt gene has been inserted into commercial cultivars R.Gala.The main results were as follows, (1) Gaining a high efficient and stable regeneration system in apple leaf in vitro.In the experiment of effects of different cytokinins on regeneration adventitious shoots from different genotype apple leaves in vitro, it were found that R.Gala was very effective on media with BA 5.0 mg/L and the best concentration of BA, CPPU and TDZ for Fuji 2001 and Nagafu No. 2 were 8.0, 8.0, 1.0-2.0 mg/L respectively, that TDZ was higher than the two other, CPPU was for first time used in the experiment of regeneration in apple and gained the better regeneration efficient. In the experiment of leaf polarity, the result showed regeneration efficient of leaf adaxial surface up was higher than one of leaf abaxial surface up, and middle leaf pieces was the highest for R.Gala, while leaf pieces of nearly petiole the highest for fuji 2001. Dark culture significantly enhanced regeneration efficiency of apple leaves in vitro, the suitable time of dark culture was different because of genotype, 21 d for Fuji2001, 14 d for R.Gala. AgNO3 was able to induce the development of mass calli tissue, which was disadvantage of shoot regeneration, but advantage ofrooting.(2) Studying effects of different antibiotic on the regeneration of apple leaves in vitro and the growth of shoots, ensured selecting pressure.It was suitable that 500 mg/L Cef and 5 mg/L Kan used acted as concentration of controlled the propagation of Ag and level of selection in the regeneration experiment of transformation in apple, that 500 mg/L Cef and 5 mg/L Kan used selected resistant shoots in the subsequent rooting experiment.(3) Establishing the genetic transformation system and transferring ipt and SSU-ipt genes into apple.In this experiments, the efficiency of exogenous gene transferring into cells of apple cultivar Royal Gala was significantly higher than cultivar Fuji 2001. The ability of strain A281-mediated gene transfer was higher remarkably than strain LBA4404. Transformation efficiency of leaf which adaxial side contacted with media during co-cultivation was three times higher than abaxial side contacted with media. Transformation efficiency had enhanced through delaying 3 d selection.Based on the regeneration and transformation systems, ipt and SSU-ipt genes changed agriculture character were transferred to apple R.Gala by Agrobacterium-mediated and gained the resistant regeneration plants, which were proved by kanamycin resistant biochemical selection and PCR. |
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