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Optimization of Transient Expression System in Chrysanthemum morifolium based on Agrobacterium Vacuum Infiltration Method |
YU Yao, CHENG Hua, CHEN Su-Mei, CHEN Fa-Di, JIANG Jia-Fu* |
State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs/Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration/College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China |
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Abstract Chrysanthemums morifolium are mostly hexaploid, with complex genetic backgrounds and low efficiency in stable genetic transformation. This study aimed to optimize the transient expression system in C. morifolium and improved the transient transformation efficiency as well as target gene expression activit, to provide an effective way to carry out functional studies of Chrysanthemum-related genes. C. morifolium 'Jinba' cuttings were transient transformed by using the vacuum infiltration method, and the infiltration efficiency was statistically analyzed by 6 aspects: Type of suspensions, pH of suspensions, whether to add acetosyringone, final concentration of Agrobacterium suspension, dark incubation time of Agrobacterium and dark incubation temperature of plants. And the expression efficiency was determined by measuring and analyzing the expression of fluorescence values of luciferase (LUC) to explore the influence degree of 6 conditions on the transient transformation efficiency and target gene expression activity of C. morifolium. The experimental results showed that 2-morpholinoethanesulphonic acid (MES) buffer was more suitable as suspension when infiltrating chrysanthemum, and the highest level of fluorescence expression was obtained when the pH of suspension was 6.0; next, the addition of acetosyringone (100 μmol/L) could improve the transformation efficiency of transient infection and the expression level of fluorescence value; the expression of fluorescence increased significantly when the final OD600 value of suspension was 1.8; the expression of fluorescence value was more favorable when the suspension was placed in dark culture at 28 ℃ for 4 h. In addition, the efficiency and the expression of fluorescence value were increased when the plants were cultured in the low temperature condition (at 10 ℃). This study significantly improved the expression of fluorescence value of C. morifolium and achieved effective transient expression in a short experimental period by changing the conditions of the composition of the suspension, the concentration of the suspension, the suspension, and the culture conditions of the plant after instantaneous transformation, which provides efficient method for the functional study of C. morifolium genes.
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Received: 07 December 2022
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Corresponding Authors:
*jiangjiafu@njau.edu.cn
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