Studies On Optimization Of The Regeneration And Genetic Transformation In Camellia Sinensis (L.) O.Kuntze

Camellia sinensis (L.) O.Kuntze is a sort of perennial evergreen tree or shrub which is classified to Theaceae, Camellia. It is one of the important economic crops in our country with a long history of cultivation. The limitations of the use of tea quality resources are the conventional breeding techniques, such as the difficulties of distant hybridization, the low beneficial mutation frequency, time-consuming on breeding of fine varieties and asynchronous flowering period. These also make the tea breeding difficult to achieve a breakthrough. In this study, an efficient plant regeneration protocol in vitro and transformation by Agrobacterium-mediated method of C. sinensis was achieved. The results were as follows:1. Establishment of high efficient regeneration system of C. sinensisThe immature embryo of C. sinensis was used as explants and a series of factors that influenced C. sinensis tissue culture were studied. The medium and hormone combinations in different culures were screened, and then the regeneration system of C. sinensis was optimized. The results indicated that:the more effective inoculation method was that the immature embryos were buried upward into the medium. The appropriate medium for callus induction was ER+0.1mg/L NAA+1mg/L6-BA+1mg/L GA3, the efficiency of induction reached98.9%; the efficiency of differentiation could be up to78.9%when the differentiation medium was ER+0.1mg/L IB A+2mg/L6-BA; The optium medium for shoots elongation was ER+0.1mg/L NAA+2mg/L6-BA+1mg/L GA3; and the most suitable rooting medium was1/2MS+0.2mg/L IBA+1mg/L KT.2. Establishment of the genetic transformation system of C. sinensis mediated by A. tumefaciensThe cotyledon and the cotyledon with immature embryo were used as receptors of Agrobacterium-mediated transformation. The factors influencing the transformation frequency were investigated, such as the infection time, the co-culture time, the co-culture medium, the concentration of acetosyringone (AS), the sub-culture time. The results indicated that infection time of15min, co-culture time of3days, YEB medium with150μmol (AS), the sub-culture time of3days could enhance the transformation frequency. The more fitting receptor for A. tumefaciens-mediated transformation of C. Sinensis was the cotyledon with immature embryo. Resistant seedlings of C. sinensis were obtained using this optimized Agrobacterium-mediated method.