Construction Of Callus Genetic Transformation System And Study On JsFLS5 Genetic Transformation Of Juglans Sigillata Dode

Walnut（Juglans sigillata Dode）,native to China,has a long cultivation history and good quality.It has high edible,economic,and ecological values.In recent years,with rapid economic development and the continuous improvement of consumption levels,the formation and regulation of walnut quality have gradually become a research hotspot.However,up to now,many limiting factors in the process of genetic transformation of walnut are still unclear,and the efficient regeneration and transformation system is still immature,which greatly limits the functional interpretation and molecular breeding process of walnut genes.Therefore,this study established a stable and efficient walnut callus genetic transformation system through induction,proliferation and maintain of callus,type and concentration of antibiotic,method and concentration of Agrobacterium-mediated.On this basis,the overexpression of Js FLS5 gene evaluated the damage degree of transgenic callus under salt stress,explored the mechanism of Js FLS5 response to salt stress,and verified the reliability of the genetic transformation system.This study laid a foundation for the function verification of key genes and the improvement of quality or stress resistance of J.sigillata through genetic engineering.The main findings are as follows:1.Establishment and optimization of callus culture and genetic transformation system of J.sigillataThe 0.5×0.5cm leaves of‘Qianhe-7’inoculated could induce 97.00%callus on the medium containing DKW+0.5 mg/L 6-BA+0.5 mg/L NAA+1.0 mg/L TDZ.In the medium of DKW+0.5 mg/L NAA+1.2 mg/L 2,4-D+0.5 mg/L IBA,the proliferation rate of callus was 516.27%.18 d was the best time for callus proliferation.Fifth callus of subgeneration was the best number of callus preservation,and the second callus of subgeneration was the most suitable for genetic transformation.The results of three different Agrobacterium transformation methods were comprehensively analyzed.It was found that the negative pressure-assisted Agrobacterium tumefaciens transformation method obtained a high genetic transformation rate（16.67%）,the lowest Agrobacterium contamination rate（0.00%）,the lowest browning rate（46.67%）,and the most suitable genetic transformation method for walnut callus.The specific system was as follows:the bacterial solution concentration was OD₆₀₀=0.6,the AS concentration was 75 mg/L,the syringe was pulled 40 times within 10 min under negative pressure treatment,the infection time was 5min,the co-culture time was 2 d,the antibiotic screening concentration was 200mg/L Kan and 500 mg/L Cef.2.Overexpression of Js FLS5 can maintain the balance of reactive oxygen species and reduce the toxicity of Na⁺to improve callus salt toleranceThe plant expression vector containing Js FLS5 was constructed and the negative pressure assisted Agrobacterium Tumefaciens genetic transformation system was used for callus transformation.The results showed that the transgenic callus showed strong green fluorescence under the microscope,the Js FLS5 gene fragment was successfully transferred into the resistant callus and overexpressed.The transgenic calli yellow fluorescence was darker and brighter than the wild type.In addition,overexpression of Js FLS5 significantly increased the content of total flavonoids,8 secondary metabolites,and 9 genes in the flavonoid biosynthesis pathway.These results indicated that Js FLS5was overexpressed successfully in the walnut’s callus,and the walnut’s genetic transformation system could be used effectively.Transgenic callus overexpressing Js FLS5 was cultured under salt stress of 100m M Na Cl to evaluate its tolerance.The results showed that the content of total flavonoids and secondary metabolites in transgenic callus increased with the increase of related gene expression,which resulted in the contents of hydrogen peroxide（H₂O₂）,malondialdehyde（MDA）,and relative electric conductivity（EL）of overexpressed calli were significantly lower than wild-type（WT）calli.In contrast,total ascorbic acid（ASA+DHA）contents,total glutathione（GSH+GSSG）in overexpressed calli were significantly higher than in wild-type calli.In addition,the Na⁺and Na⁺/K⁺ratio decreased significantly in transgenic callus under salt stress.These results indicate that transgenic calli can effectively improve the reactive oxygen species balance and reduce the toxicity of Na⁺by regulating the synthesis of flavonoids,which improving the basic tolerance of J.sigillata to salt stress.