The Establishment Of Genetic Transformation In Walnut(Juglans Regia.L) And The Study On Gene Transduction Between Grafts

Walnut(Juglans regia.L)is a kind of tree species with important edible value and economic application value.It is of great significance to cultivate rootstocks with dwarfing characteristics and enhanced resistance because of its tall tree,habitat mostly mountain,long reproductive cycle and other limiting factors.In this study,the RNA interference vectors constructed by GA20 ox gene in Carya cathayensis ware transformed into walnut somatic embryos by agrobacterium mediated method which provide a feasible method for breeding dwarfing rootstocks.Studying on transgene signal transduction between rootstock and scion by DsRED transgenic walnut seedlings which can lay the foundation for the study of the transfer mechanism of functional genes in grafting and provide a platform for further study on the molecular mechanism of gene transfer.The results were as follows:(1)The establishment of genetic transformation system: DKW medium as the basic culture medium of walnut somatic embryo culture,fully able to meet the RR4 walnut somatic embryos of the normal growth and reproduction,every 7-10 days for a subculture,to maintain the best growth of somatic embryos;The somatic embryos occurred directly on the surface of the embryos and the cotyledon of the zygotic embryos.The somatic embryos developed four stages: spherical embryo,heart embryo,torpedo embryo and cotyledon;Agrobacterium tumefaciens concentration OD600 was 0.4 infection embryos 20 min and OD600 was 0.5 infected 15 min can get the better results.After the gus and qRT-PCR test,the positive somatic embryos and seedlings were obtained,and the expression of GA20 ox gene in somatic embryos and plants decreased significantly compared with the control.It is clear that the transformants are shorter than the control plants.(2)The establishment of in vitro grafting system: Fluorescence microscopy showed that the DsRED transgenic seedlings showed red fluorescence and the control did not show fluorescence.After screening for one month,the transgenic seedlings grown normally,while the control seedlings slowly darkened.PCR showed that the DsRED transgenic plantlets had high expression level and stable expression but there were no gene expression in the control seedlings.In the in vitro grafting test,the callus healing between the rootstocks and the scion were healed after two weeks.The results of qRT-PCR showed that the DsRED in the transgenic stocks were detected in the non-transgenic scion,but under the fluorescence microscope can not be observed the bright red fluorescence.