Studies On Agrobacterium-tumefaciens Mediated Genetic Transformation Of Friable Embryogenic Calli By AtWUS And AtMYB118Genes In Hevea Brasiliensis

The friable and embryogenic calli of rubber tree grow fast, and can be subcultured for long time, then regeneration plantlets can be obtained. So these calli are good materials for genetic transformation. In this study, the stem cell determinable gene WUS from Arabidopsis thaliana was cloned into plant expression vector. The factors including kanamycin concentration, preculture time, AS concentration, Agrobacterium concentration, infecting time, co-cultivation time and co-cultivation temperature during genetic transformation procedure were optimized by orthogonal design method. The friable embryogenic calli of rubber tree elite clone Reyan8-79and Reyan88-13were used as genetic transformation recipient materials, after5-day cocultivation, these calli were placed on screening medium which contains75mg/L or100mg/L kanamycin.4-6months later, a lot of resistant friable calli were obtained. All of these friable calli, after histochemical detection for GUS activity, were transferred into the maintenance medium for proliferation about1-2months, which contains75mg/L or100mg/L kanamycin, then were induced embryoids and regeneration plantlets. By means of testing GUS activity, PCR detection, sequencing and southern blotting, it showed that WUS gene had been integrated into the genomic DNA of rubber tree, and some phenotype characters could be observed, such as many protuberances growing near the base of cotyledon, adventitious buds emerging, et al. The main results are the following:1) Cloning of WUS gene in Arabidopsis thaliana. The specific primer were designed based on ORF of WUS gene which accession number is in NCBI. After extraction of the total RNA of Arabidopsis thaliana by Trizol method, a DNA fragment about900bp was obtained through RT-PCR. DNA fragment sequencing confirmed that it was AtWUS gene. So a full length AtWUS gene was cloned.2) The studies on the effects of factors on Rubber tree fraible embryogenic calli genetic transformation. Based on UidA activity and calli survival rate, orthogonal design was carried out, by which all of the factors about transformation were optimized. The suitable results were the following:pre-culture time was0day, bacterial concentration OD600was0.7, AS concentration was200μM, co-cultivation time was5days, co-cultivation temperature was25℃, and infecting time was7min.3) Due to the resistant response difference of different clones to kanamycin and the growth rate difference of calli, suitable concentrations of kanamycin for screening rubber tree clone Reyan8-79and Reyan88-13resistant calli were determined, which were75mg/L and100-125mg/L respectively.4) Resistant embryogenic calli, embryoids and plantlets were obtained. After co-cultivation for5days, rubber tree clone Reyan8-79and Reyan88-13calli were transferred into decontamination medium for18days then transferred into maintenance media containing75mg/L and100-125mg/L kanamycin for4-6months respectively. Later some yellow calli emerged. When they grew up, a part of them were used for GUS staining, other parts were proliferated for about1-2months then were used for inducing embryoids and plantlets.17resistant callus lines and1539resistant embryoids were obtained from Reyan8-79, meanwhile,5resistant callus lines,843embryoids and21putative transgenic plantlets were obtained from Reyan88-13,26putative transgenic plantlets were obtained from Reyan8-79.5) Drawing a comparison of the numbers of resistant embryoids with those of negative control embryoids, the difference was not notable. Thus it suggested that the AtWUS gene driven by only one CaMV35S promoter might not promote somatic embryogenesis in transgenic calli.6) Molecular analysis. Out of21resistant plantlets of Reyan88-13clone,7were chosen randomly and used for molecular analysis with3normal test tube plantlets as negative control. In which2plantlets were proved to be transgenic plantlets.7) Plantlets were used for GUS stainning. Other14resistant plantlets of Reyan88-13clone were analyzed by GUS stainning by cutting their cotyledons.6plantlets were positive for GUS detection, the rest were negative. The positive performance of some of the positive plantlets were notable. At the same time,1of26plantlets was positive by their leaves, root or part of embryoid GUS detection.8) In this study, orthogonal design method was adopted to optimize the genetic transformation techniques using friable embryogenic calli as recipient materials in Hevea brasiliensis. Transgenic plantlets had been obtained. So it was beneficial to rubber tree clone genetic amelioration.