Study On Agrobacterium-mediated Genetic Transformationof Maize Immature Embryos

ObjectiveThis study used the key enzyme gene of sorghum HCN approach,CYP71E1,as the target gene,and the maize immature embryo as an explant,and carried out a study on the genetic transformation of maize through the mediation of agrobacterium tumefaciens.The A188 maize regeneration system was optimized,and how different explant size,concentration of agrobacterium tumefaciens,infection time and heat treatment time will influence the efficiency of genetic transformation of maize had been analyzed,simultaneously,the transgenic plant was identified by the use of GUS histochemical staining and PCR technology.The highly-efficient conversion technology was used to solve the problem of bottleneck that threatens the yield and quality of maize brought by biotic and abiotic stress,providing a theoretical basis for the transformation of new variety of maize.MethodsBy the polymerase chain reaction(PCR),a 1854 bp full length gene of CYP71E1 was amplified from the sorghum.By the use of Gateway technique based on the site-specific recombination,the CYP71E1 gene was recombined into the expression vector(p MDC141),the successfully-constructed expression vector CYP71E1-p MDC141 was imported into the agrobacterium tumefaciens EHA105 bacterial strain.The maize regeneration system was established through an optimization of explant material,healing-induced culture medium,differential medium and root medium.The immature embryo of maize elite inbred line A188 was infected by the EHA105 bacterial strain of agrobacterium tumefaciens that carried the CYP71E1-p MDC141(containing GUS gene)Plasmid,the mean efficiency of conversion of GUS gene was improved by an optimization of explant size,concentration of agrobacterium tumefaciens,infection time and heat treatment time,all of which had an influence on the agrobacterium tumefaciens-mediated transformation of maize immature embryo.Meanwhile,the transgenic plant underwent a PCR and GUS detection.ResultsBy the use of Gateway technique based on site-specific recombination,the key enzyme gene CYP71E1 of HCN approach of sorghum was cloned to the entry vector p CRTM8/GW/TOPO?through TOPO clone,the entry vector was cloned and recombined into the expression vector(p MDC141)with the help of LR clone enzyme,the plant expression vector p MDC141-CYP71E1 was established successfully,which was then imported into the agrobacterium tumefaciens EHA105.A highly-efficient maize regeneration system was established,it was found that the callus initiation rate was the highest when the maize immature embryo was 1.0~1.2mm in size,the embryogenic callus culture medium was induced and optimized by means of orthogonal experiment.It was then concluded that,the optimal combination of addition of the three factors,L-pro?2,4-D?Ag NO3,into the MS basal culture medium that induces the callus tissue of maize A188 was : L-pro 700 mg/L?2,4-D 2.0 mg/L?Ag NO3 8 mg/L.it was found that,the differential efficiency reached its peak when the zeatin concentration was 5mg/L in the differential medium.The optimal concentration of IBA in the root medium was 2mg/L.Establishment of a highly-efficient genetic transformation system,the mean GUS transformation rate was 35.3% when the maize immature embryo was1.0 ~ 1.2mm in size;it was 46.8% when the concentration of bacterial suspension of agrobacterium tumefaciens OD660 nm was 0.8;it was 46.4% and45.4% when the infection time was 5 min and when it was pre-heated for 3 min under 43 ?.Thus,the optimal transformation condition: selecting the immature embryo that is 1-1.2mm in size;bacterial suspension concentration OD660nmfor0.8;infection time for5 min;preheating for 3 min under 43 ?.12 transgenic seedlings had been obtained from the PCR and GUS detection of transgenic plant that was acquired.ConclusionsCompared with the traditional clone methods,Gateway technique is faster,more convenient and has a higher positive cloning efficiency,the expression vector p MDC141-CYP71E1 can be successfully established with Gateway technique.A188 maize immature embryo regeneration system has been established through an optimization of explant material,callus inducing culture medium,differential medium and rooting medium.Through an optimization of part of the factors that influence the agrobacterium tumefaciens-mediated maize immature embryo,the optimal transformation condition has been determined as: selecting the immature embryo that is 1.0 ~ 1.2mm in size;bacterial suspension concentration OD660nm=0.8;infection time =5 min;preheating for 3 min under 43 ?.A positive plant with key enzyme gene CYP71E1 of sorghum HCN approach has been obtained.