Study On Genetic Transformation Of Maize Somatic Embryogenesis System

Maiz（eZea mays L.）is one of the world’s leading crops, whether in agriculture orindustry the needs of which are increasing. Molecular Breeding plays an importantrole in Maize Breeding. As to transgenic maize breeding, it is well known that toestablish the genetic transformation system is of the great importance A large numberof experiments show that thesomatic embryogenesis system induced by immatureembryos has many advantages such as bipolarity, genetic stability and so on.Therefore,the somatic embryogenesis system is preferred in maize genetictransformation technique. However, it is very difficult to establish a good and stablemaize somatic embryogenesis system. Consequently,we performed the followingresearches on the establishment of the maize somatic embryogenesis system andthetransformation of the related genes.In this study, the maize immature embryoswere obtained from Y423,an eliteinbred line of China, from which somatic embryos were induced and the geneZm22Arelatingto embryogenesiswas cloned Based on the somatic embryogenesissystem, conditions of Agrobacterium infection and regeneratestransplanting wereoptimized. The system was further used in the study of genetic transformation ofgenesZm22A and ZmSh2. The main findings are as the followings:We cloned the maize embryogenesis-related geneZm22A by RT-PCR. Thesequencing results showed that: the full-length of the Zm22A was1317bp, encoding438amino acids. Homology analysis revealed that the Zm22Agene encodedtheGTPase activating protein,which molecular weight is50,289and isoelectric pointis5.70. Meanwhile, we analysedthe Zm22A amino acid sequence by bioinformatics.The results showed that: compared with rice, millet,etc,the homology of amino acidsequence reached above80%. So we deemed thisgeneas embryogenesis-related gene.Using bioinformatics software wefurtherpredicted its biological and structuralproperties.All the results proved that we got the full length ofgene Zm22Acorrectly. In this study, the condition of Agrobacterium infected embryos were explored.We set up different infection under0.09MPa respectivelysuch as5min,10min,15min.Then we shaked the infected embryos at28℃,180rpmfor20min. The GUStransient expression and the resistant callus statistical results showed that infectionunder-0.09MPa for5min wasthe best one. Regenerated plants from Y423somaticembryos were used to study the impact of time for hardening plantlets on survival rateof transplanting.The results indicated that regenerated plants hardened for4d, hadthehighestsurvival rate of transplanting, reaching91%.This study successfully constructed the plant expression vector35S::Zm22A.Immature embryos from an inbred line of maize, were transformed by AgrobacteriumEH105. Then the infected embryos were Selected at the screening medium containing1.5mg/L bialaphos to obtain resistant callus and at last,the resistant regenerant wereplanted. The results showed that the differentiation rate of resistant callus with targetgene was improved.We also used the systemto carry out the genetic transformation of large-subunitSh2inmaize AGPase.423plants which resistant Bialaphos were obtained, and afterfurther tested60positive plants were obtainedby PCR.43with Bar,37with RP5,andtarget gene（Sh2）.We cloned the embryogenesis-related gene, Zm22Afrom the maize somaticembryogenesisandconstructed its expression vectorsuccessfully.Moreover, weoptimized the system of Agrobacterium infected somatic embryogenesis, andapplied itto transform two maizegenes Zm22A an ZmSh2. Our work will proved importantinformation for the maize genetic breeding...