Agrobacterium-Mediated Genetic Transformation Of Walnut(Juglans Regia L.)

With the development of modern molecular biology,plant transgenic technology plays an important role in the study of gene function and the breeding of transgenic new varieties.Agrobacterium-mediated transformation has been used in the genetic modification of plants to enhance their tolerance to biological and abiotic stresses,improve nutrient capture and insect resistance,improve crop productivity and reduce the use of harmful pesticides.In this study,the conditions of induction of somatic embryos from young embryos and the maturation and germination of somatic embryos were optimized.JrWRKY4 gene was transferred into walnut somatic embryo by agrobacterium-mediated method.The optimal conditions of the regeneration system and genetic transformation system of walnut somatic embryo were studied,which laid a foundation for the analysis of walnut gene function and transgenic breeding.The main results are as follows:1.In this experiment,cotyledons of young embryo of walnut were used as explants to induce somatic embryo differentiation and maturation and obtain regenerated plants.Through morphological and cytological observation of somatic embryo formation and development,the structural changes of somatic embryo development in walnut were revealed in the spherical stage,heart-shaped stage,torpedo shaped stage and cotyledon stage,and the polar development process of somatic embryo was obviously observed,which was similar to the formation process of zygotic embryo.During the spherical phase,the inner layer cells were observed to divide into the basic vascular tissue,the primordial cambium was developed in the torpedo phase,and the gradual establishment of polarity was clearly observed.During the cotyledon phase,Y-shaped vascular bundles were clearly observed.2.Construction of Agrobacterium-mediated walnut somatic embryo transgenic system:selecting untransformed walnut somatic embryos with good growth,in order to achieve the screening effect,genetically transformed antibiotics are selected to be kanamycin with a pressure of 50 mg·L^-1;Optimized somatic embryo transgenic conditions,Agrobacterium solution concentration OD600=0.6,infection time 20min,co-cultivation for 1 d,bacteriostatic carbenicillin concentration 250 mg·L^-1,PRI101-JrWRKY4 transferred into walnut somatic.3.The somatic embryos screened by Kana resistance can be amplified by PCR to detect a band of about 1554bp;qRT-PCR detection of the expression level of JrWRKY4 gene in transgenic somatic embryos,the expression level of JrWRKY4 gene in walnut transgenic somatic embryos Compared with untransformed somatic embryos,the expression level of JrWRKY4 gene in transgenic somatic embryos is nearly 10 times that of untransformed somatic embryos;Western Blot detection of transformed somatic embryos can detect the purpose of about 90KD Bands.