Transformation Acceptor System Optimization And Transient Expression Of APX Gene In Apple

Apple(Malus domestica) is one of the main cultivated trees in deciduous fruit. It is very difficult to be improved through cross breeding regularly. So it is an important means to get transgenic plant via gene transfered technique in apple breeding.The apple's leaf is an ideal acceptor material for transformation. Because of it's lower regeneration frequency and conversion, the development and application were restricted in apple transformation technique.Ascorbate peroxidase (APX) is critical for protection of cellular components against oxidative injury. The activity of APX can be increased by transformation technique and enhanced the ability of reactive oxygen species(ROS) scavenger enzyme in plants. Therefore it palys a significance role in improving the ability of oxidation force of plants. In this article, factors affecting buds rengeneration from apple leaves were studied. Regeneration system of Gala and NagafuNo.2 leaves in vitro was optimized. By studying factors affecting Agrobacteium-mediated transformation, the Agrobacteium-mediated transformation system was established by means of gus and gfp gene transient expression and leaf dises.Main results of this study are as follows:1.High efficient and steady regeneration system of apple leaf in vitro was obtained. By adjusting the factors such as concentrationof growth hormone, dark incubation period, leaves from different parts and leaf polarity,established high efficient regeneration system of Gala and NagafuNo.2. On MS medium congtaining TDZ 0.5 mg·L-1 + NAA 0.4 mg·L-1 or TDZ 4.0 mg·L-1 + NAA 0.6 mg·L-1 with abaxial surface of Gala lwaves, adaixial surface of NagafuNo.2 leaves touching medium and after about 21d dark incubation treatment, the regeneration efficiency of Gala and NagafuNo.2 leaves reached 100% and 67%,respectively.2.The genetic transformation system was established via the transient expression of APX gene.Concentration of antibiotics was ensured. By optimizing such factors which may affect transformation as pre-culture, co-cultivation period, concentration of agrobacteium used for infection and infection period, a high-efficient genetic transformation system by means of APX gene transient expression.The result showed that the suiltable conditions of transformation conteined 5 mg·L-1 Kan, 200 mg·L-1 Cef, 2d preculture,3d coculture , 5min infection, 0.4 OD600. It has a high gus/gfp transient expression frequency. Delaying selection after coculture is favourable to the transformation.