Study Of The ACC Oxidase Transplantation System Of Jiro And Uenishiwase

In this study,transformation systems had been established through selecting the best conduction of regeneration,transformation and antibiotic selection of Jiro and Uenishiwase leaves in vitro,and ACC oxidase RNAi gene had been inserted into Jiro and Uenishiwase. We can increase the storaging function of climacteric fruit by repressing the expression of the ACC oxidase enzymes with transgenetic method.The main results were as follows:1.Establishing a high efficient and stable genetic transformation system of Jiro in vitro.1.1 Studying effects of antibiotic on the regeneration of Jiro leaves in vitro and the proper concentration of antibiotic had been found.Studying effects of antibiotic on the regeneration of Jiro leaves and leaf explants, ensured the proper concentration of antibiotic.Using 200 mg/L Cef and 30 mg/L Sp on growth of resistant shoots.1.2 The transgenic technic of JiroCutting 4mm×4mm from the base of 2 to 3 leaves of the top of Jiro of 4.5 years in vitro from generation to generation,putting with top of the back of leaf on the medium of DKW(1/2N)+ZT 1.0 mg/L+TDZ 0.5 mg/L,dipping the leaves after pre-cultured in darkness for four days into Agrobacterium suspension which was AS 50 mg/L in it for ten minutes,transferring the leaves to the medium of DKW(1/2N)+ZT 1.0 mg/L+TDZ 0.5 mg/L with co-cultured in darkness for three days,then diverting the leaves to the medium of DKW(1/2N)+ZT 1.0 mg/L+TDZ 0.5 mg/L+Cef 200 mg/L with de-cultured in darkness for three days,finally transferring the leaves to the medium of DKW(1/2N)+ZT 1.0 mg/L+TDZ 0.5 mg/L+Cef 200 mg/L+Sp 30 mg/L for selecting in light,once every twenty-five days,transferring the leaves to the medium of DKW+ZT 1.0 mg/L+Sp 30 mg/L+Cef 200 mg/L after seven to eight times.In this study,we obtained twenty-seven Jiro transgenetic plants carrying ACC oxidase RNAi gene which identified by histochemical analysis of GUS activity and PCR.2.Establishing a high efficient and stable genetic transformation system of Uenishiwase in vitro. 2.1 Studying effects of antibiotic on the regeneration of Uenishiwase leaves in vitro and the proper concentration of antibiotic had been found.Studying effects of antibiotic on the regeneration of Uenishiwase leaves and leaf explants,ensured the proper concentration of antibiotic.Using 200 mg/L Cef and 30 mg/L Sp on growth of resistant shoots.2.2 The transgenic technic of UenishiwaseCutting 4mm×4 mm from the base of 2 to 3 leaves of the top of Uenishiwase of 4.5 years in vitro from generation to generation,putting with top of the back of leaf on the medium of DKW(1/2N)+ZT 1.0 mg/L+TDZ 0.5 mg/L,dipping the leaves after pre-cultured in darkness for three days into Agrobacterium suspension which was AS 100 mg/L in it for ten minutes,transferring the leaves to the medium of DKW(1/2N)+ZT1.0 mg/L+TDZ 0.5 mg/L with co-cultured in darkness for four days,then diverting the leaves to the medium of DKW(1/2N)+ZT 1.0 mg/L+TDZ 0.5 mg/L+Cef 200 mg/L with de-cultured in darkness for three days,finally transferring the leaves to the medium of DKW(1/2N)+ZT 1.0 mg/L+TDZ 0.5 mg/L+Cef 200 mg/L+Sp 30 mg/L for selecting in light,once every twenty-five days,transferring the leaves to the medium of DKW+ZT 1.0 mg/L+Sp 30 mg/L+Cef 200 mg/L after seven to eight times.In this study,we obtained four Uenishiwase transgenetic plants carrying ACC oxidase RNAi gene which identified by histochemical analysis of GUS activity and PCR.