Preliminary Studies On Pollen-mediated And Agrobacterium-mediated Genetic Transformation Of Fruit Trees

The homologous genes, AFL1 and AFL2, which were cloned from apple (Malus pumila Mill) by Dr. Cao Qiufen in 2003, were used to transform apple cultivar Gana and grape {Vitis vinifera L) cultivars, Meiguixing and Guibao, using the methods of pollen-mediated plus ultrasonic treated and agrobacteria-mediated transformation to study the feasibility of pollen-mediated genetic transformation in fruit trees. Factors affecting transformation of apple and grape were also studied. Basic parameters such as buffers suitable for pollen germination and ultrasonic treatments of apple and grape pollen were determined. Moreover, factors affecting agrobacteria-mediated genetic transformation of in vitro apple leave pieces were studied and regeneration system for transformed plants from apple cultivar Gala was established.The results are as follows:1. The buffer pH value suitable for apple pollen treatment was 5.4 and sucrose concentration, 10%, in the ultrasonic treatment of pollen when using pollen-mediated genetic transformation. The ultrasonic power, treatment time and times had a significant effects on the pollen germination rate and broken rate. The GUS activity examination showed that the pollen transformation rate was 8%.2. Factors such as power of ultrasonic waves, treatment of time and times etc. also had strong effects on grape pollen and interactions existed between factors. The significance test showed that 100-120W of ultrasonic power, 4-5s of treatment and 2-6s of gap between treatments and 6 times of treatments were the best combination for grape pollen transformation, with the 0.4% of pollen transformation rate by GUS activity test. Selected parameters of ultrasonic treatments were used to transform grape, 578 cross seedling were obtained.3. The effects of hormone in medium, dark culture and ways of leave pieces laying up on plant regeneration from apple cultivar Gala were examined and efficient plant regeneration system was established. Adventitious buds and plants ware regenerated from in vitro leave pieces of apple cultivar Gala cultured in MS medium with 5mg/L of BA and 0.5mg/L of IAA and leave adxial surface touching medium. The plant regenerating rate reached 97%.4. The effects of different antibiotic on adventitious buds were studied to optimize the selection pressure. In the transformation system of Gala, 400mg/L of Cef and 5mg/L of Kan were added to as the suitable selection pressure in de-bacteria culture. Genetic transformed calli and resistant plants were obtained.