A Genetic Transformation System For Sweet Cherry(Prunus Avium L.) Shoot Tip Mediated By Agrobacterium Tumefaciens

Xylophyta, including most fruit trees and all forest-trees, play an important role in national economy and in maintenance and preservation of the ecosystem. In contrast to herbaceous plant, the long juvenile period is the main obstacle to traditional breeding of trees. The long time between seed germination and flowering, prevents rapid detection of many tree traits, such as the traits of fruit and seed, till the tree reaches maturity. Moreover, the difficulties in parents selection and mating control add to the limitations of tree breeding. Finally, the size of trees and the area required for field trails create considerable difficulties in assessing the performance. The technology of plant genetic transformation provides a efficient method to overcome many of these obstacles and to accelerate tree-breeding programs. During the last twenty years, the technology of plant genetic transformation has experienced a great advance and vast application. But the genetic transformation of xylophyta is far lagged behind that of most agronomic species, mainly because lack of a high efficient system of regeneration and genetic transformation.Cherry, with popular fragrance and taste, is the earliest spring fruit. But cherry has small fruit, large stone, thin flesh and is easy to produce dehiscent fruit. As a xylophyta, traditional improvement of cherry is very difficulty. In this thesis, Using the shoot tip, a high efficient regeneration system of cherry was constructed, and using the domesticated shoot tip as receptor, the A. tumefaciens mediated genetic transformationsystem of cherry was optimized by comparing main parameters of the A. (umefaciens mediated genetic transformation. Main results are as following.1. Regeneration system of sweet cherry shootBy comparing the efficiency of different hormonal composition, the optimal regeneration system of cherry was determined: differentiation medium, B5+1.0mg/LBA+ 0.1mg/LNAA , B5+1.0mg/LBA+0.2~0.5mg/LNAA; proliferation medium, B5+BA0.25mg/L+NAA0.05; and rooting medium. B5+ IBA0.2-0.5 mg/L. This regeneration system had a high shoot-initiated regeneration frequency of 97%.2. It was determined that the inhabiting concentration of Cef was 500mg/L, and the selection concentration of kan was 50 mg/L.3. Shoot tip transformation system mediated by A. tumefaciensUsing domesticated shoot tip as receptor, the parameters of A. tumefaciens transformation was optimized. The optimized condition was that the concentration of bacterium suspension for infection was 0.5 OD600, infecting period was 30 min, co-culture time was 3d. With these parameters, the percentage of green bud increased to 0.78%.4. The green bud efficiency of immediate selection, delaying selection and gradient selection was compared, suggesting that gradient selection had a highest efficiency of 0.85%.5. It was found that 120mmol / L AS, 0.1% Tween20 and 1min X 10 of negative pressure had optimum effect on increasing the green bud efficiency.6. Using the optimized transformation system, a plant expression vector pIG121 was introduced into sweet cherry. PCR analysis of resistant seedlings with NPTII gene primers showed that 6 out of 12 seedlings detected had the 700bp fragment specific to the plasmid pIG121, indicating that T-DNA had been integrated into the genome of sweet cherry.