Studies On Polyphenol-resisting Characteristics Of Several Kinds Of Agrobacterium And The Demestication Of Polyphenol-resisting Strain

The Camellia sinensis(L.) Kuntze is one of the major cash crops,and the tea industry takes an important place in agriculture industries in China.The current market demand of tea products is changing and the traditional breeding can't meet the demands of tea production because the cycle of the traditional breeding takes decades of identification period.With the development and mature of biochemistry and transformation technologies, plant gene engineering can be used in directional genetic breeding of tea plant[Camellia sinensis(L.) Kuntze].The Agrobacterium-mediated genetic transformation was widely used in plant transformation.It was also used in tea genetic transformation but also need more studies.It was reported that hairy root and some GUS transient expression were got,but the inducing rate was between 4%-25%.Tea plant(Camellia sinensis) was multi-year growth wood and plenty of polyphenol,and it was the key factor of inducing the hairy root forming.The result was not clear enough now,so this paper chose two kinds of Agrobacterium rhizogenes and Agrobacterium tumefaciens to make clear the variation of polyphenol-resisting characteristics of different kinds of Agrobacterium and the polyphenol-resisting demestication of Agrobacterium strains and the rate of transient expression of GUS gene induced by the domesticated strain in callus and leaf explants.It is suggested to know more about the way of tea polyphenol inducing Agrobacterium-mediated genetic transformationin order to improve the rate of genetic breeding of tea plant.In order to make clear the variation of polyphenol-resisting characteristics of different kinds of Agrobacterium as well as the potential of improving the polyphenol-resisting ability and the Agrobacterium-mediated genetic transformation rate by domestication culture,the inhibition effect of tea polyphenols on four kinds of Agrobacterium strains,the polyphenol-resisting demestication of Agrobacterium strains and the genetic transformation ability improvement of demesticated polyphenol-resisting strain were studied.The results showed that tea polyphenols had obvious inhibition to the growth of different kinds of Agrobacterium,but it varied greatly among those Agrobacterium we studied.Some factors which affect the result of Agrobacterium tumefaciens infection on leaf and callus of teaplants were studied based on GUS transient expression.50 mg/L kanamycin is suitable selection concentration for expression vectors with neomycin phosphotransfe rase gene(NPTⅡ)as callus formation from tea plant leaf was depressed by about 30 mg/L kanamycin.The suitable system of Agrobacterium tumefaciens-mediated transformation is suggested that tea plant leaf was infected 10 min by EHA105 of OD600 about 0.5-0.8 followed by co-culture of 3 days about 28℃in darkness.A polyphenol-resisting strain EHA105AP was obtained by domestication culture procedure,the polyphenol-resisting ability of EHA105AP had the distinct enhancement compared to the primitive strain EHA105,which enhanced 90.89%compared with that of the original.Meanwhile,the rate of transient expression of GUS gene induced by the domesticated strain increased 43.85%and 57.55%respectively in callus and leaf explants case,and reached the remarkable difference(P＜0.05).