Study On In Vitro Efficient Regeneration System And Genetic Transformation Of Apple Self-crossed Progeny From ‘Megumi’

For screening the higher regeneration frequency genotype of in vitro apple leaf and providing new materials for the study of transgenic apple, Meg-5 was selected as the strongest proliferation individual plant in this experiment among the self-fertilized F1 generation of ’Megumi’ and the hybrid F1 generation of ’Huafu’ × ’Royal Gala’, ’Hanfu’ × ’Delicious 80-3’, ’Delicious 80-3’ × ’Hanfu’. The major factors effecting it′s adventitious bud regeneration were studied and Meg-5 leaf high efficient regeneration system in vitro were established. Further more, the genetic transformation system of Meg-5 mediated by Agrobacterium tumefaciens were preliminarily established. The main study contents and results were as follows:1. Proliferation ability of the in vitro plantlet of the progenies from different combinationsThere were significantly differences in the proliferation ability of the in vitro plantlet from different parents. Progenies of ’Huafu’ × ’Royal Gala’ have stronger proliferation ability, and the strongest plantlet was 09-294, it′s breeding multiple was 3.06. The proliferation ability of the generations from ’Hanfu’ × ’Delicious 80-3’ and ’Delicious 80-3’ × ’Hanfu’ were all weak, their breeding multiples were 1.03~1.56.2. Study on the in vitro high efficient regeneration system of Meg-5 leafThe relative factors(different hormone concentration, incubation time, leaf position, leaf laying methods and in vitro plantlet age) were studied for their effect on Meg-5 in vitro leaf regeneration. The results showed that the appropriate regeneration medium formula was MS+2.0 mg/L TDZ+0.5 mg/L NAA+30 g/L sucrose+2.6 g/L gelzan, dark incubation time should be controlled within 14~21d, the regeneration ability increased from the tip to petioles, leaf back downward was superior to leaf back upward,and the highest regeneration efficiency was observed at the in vitro plantlet age of 20~30 d. With the above condition, the in vitro regeneration frequency of Meg-5 leaves could be 100% and the average number of regeneration bud was 31.59 maximum.When adventitious bud was more than 3 cm long, the plantlet could be transferred to the rooting medium(1/2MS+0.3 mg/L IBA+0.2 mg/L IAA+30 g/L sucrose+6.5 g/L agar), and the rooting rate was 100%, the average rooting number was 7.26, the average rooting long was 3.9 cm. The survival rate of seedling transplanting was 100%, and the whole plant was obtained.3. Study on the genetic transformation system of Meg-5 mediated by Agrobacterium tumefaciensOn the basis of Meg-5 in vitro leaf efficient regeneration system, the effect of the antibiotic concentration, agrobacterium bacteria concentration, pre-culture time, microbial infection time, co-culture time, screening delay time on the transformation efficiency were also studied. The results showed that appropriate screening pressure Km was 15 mg/L, the concentration of the Cef was 200 mg/L, microbial concentration OD600 was 0.4~0.6, infection time was 7 min, pre-culture time was 2 d, co-culture time was 2~3 d, screening delay time was 2 d. The transformation efficiency of Meg-5 was the highest in this system, and the rate of resistant buds was 4.17% maximum.Random selection of resistant buds were detected by PCR, the transgenic plants of Meg-5 can be successfully obtained by using this transformation system.