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
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.
[1] 安文杰, 张永侠, 原海燕. 2020. 红籽鸢尾瞬时表达体系的建立[J]. 分子植物育种, 18(19): 6359-6363. (An W J, Zhang Y X, Yuan H Y.2020. A system establishment of transient expression on Iris foetidissima L.[J]. Molecular Plant Breeding, 18(19): 6359-6363.) [2] 邓叶, 阳淑金, 杜新平, 等. 2017. 菊花高效瞬时转化体系建立及稳定遗传植株再生[J]. 南京农业大学学报, 40(01): 48-53. (Deng Y, Yang S J, Du X P, et al.2017. Establishment of efficient transient transformation system and the regeneration of stable transformants in chrysanthemum[J]. Journal of Nanjing Agricultural University, 40(01): 48-53.) [3] 邓艺, 曾炳山, 赵思东, 等. 2010. 乙酰丁香酮在农杆菌介导的遗传转化中的作用机制及应用[J]. 安徽农业科学, 38(05): 2229-2232. (Deng Y, Zeng B S, Zhao S D, et al.2010. Mechanism and application of acetosyringone in Agrobacterium transformation[J]. Journal of Anhui Agricultural Sciences, 38(05): 2229-2232.) [4] 霍琳, 及晓宇, 王玉成. 2016. 农杆菌介导的烟草瞬时表达试验条件优化[J]. 分子植物育种, 14(01): 80-85. (Huo L, Ji X Y, Wang Y C.2016. Transient expression conditions of tobacco transformation mediated by Agrobacterium[J]. Molecular Plant Breeding, 14(01): 80-85.) [5] 贾红梅, 王碧玉, 刘迪, 等. 2017. 农杆菌介导CBL基因对菊花品种'c008'的转化[J]. 西北林学院学报, 32(01): 184-189. (Jia H M, Wang B Y, Liu D, et al.Agrobacterium-mediated transformation of Chrysanthemum morifolium 'C008' with CBL gene[J]. Journal of Northwest Forestry University, 2017, 32(01): 184-189.) [6] 李刚, 宋平丽, 王翔, 等. 2021. 农杆菌介导的杜梨叶片瞬时转化方法的建立[J]. 果树学报, 38(11): 2006-2013. (Li G, Song P L, Wang X, et al.2021. Establishment of Agrobacterium tumefaciens mediated transient transformation system in young leaves of Duli pear (Pyrus betulifolia)[J]. Journal of Fruit Science, 38(11): 2006-2013.) [7] 李静, 陈敏, 刘现伟, 等. 2006. 莴苣高效瞬时表达体系的建立[J]. 园艺学报(02): 405-407. (Li J, Chen M, Liu X W, et al.2006. A highly efficient system establishment of transient expression in lettuce[J]. Acta Horticulturae Sinica, 45(02): 405-407.) [8] 李辛雷, 陈发棣. 2007. 菊花自交不亲和性初步研究[J]. 武汉植物学研究(06): 591-595. (Li X L, Chen F D.2007. Self-incompatibility in Dendranthema morifolium[J]. Journal of Wuhan Botanical Research, 25(06): 591-595.) [9] 孙春莲, 王洪洋, 田振东. 2015. 农杆菌介导的pCB302-3载体在本氏烟中瞬时表达条件优化[J]. 华中农业大学学报, 34(03): 5. (Sun C L, Wang H Y, Tian Z D.2015. Optimization of Agrobacterium-mediated transient expression conditions of the pCB302-3 vector in Nicotiana benthamiana[J]. Journal of Huazhong Agricultural University, 34(03): 8-12.) [10] 张悦婧, 李颖, 王娟娟, 等. 2022. 不同转化条件对3种农杆菌GFP基因在本氏烟草中瞬时表达的影响[J]. 植物研究, 42(01): 121-129. (Zhang Y J, Li Y, Wang J J, et al.2022. Effects of three kinds of Agrobacterium and different transformation conditions on the transient expression of GFP in Nicotiana benthamiana[J]. Bulletin of Botanical Research, 42(01): 121-129.) [11] 周丹丹, 俞嘉宁. 2013. 植物细胞中瞬时表达系统的建立及研究进展[J]. 中国农学通报, 29(24): 151-156. (Zhou D D, Yu J N.2013. The progress of establishing transient expression system in plant cell[J]. Chinese Agricultural Science Bulletin, 29(24): 151-156.) [12] Bruun-Rasmussen M, Madsen C T, Jessing S, et al.2007. Stability of Barley stripe mosaic virus-induced gene silencing in barley[J]. Molecular Plant-Microbe Interactions, 20(11): 1323-1331. [13] Cakir C, Scofield S R.2008. Evaluating the ability of the Barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes[J]. Cereal Research Communications, 36(Suppl 6): 217-222. [14] Hong B, Tong Z, Ma N, et al.2006. Expression of the Arabidopsis DREB1A gene in transgenic chrysanthemum enhances tolerance to low temperature[J]. The Journal of Horticultural Science and Biotechnology, 81(6): 1002-1008. [15] Huang Y, Xing X, Tang Y, et al.2022. An ethylene-responsive transcription factor and a flowering locus KH domain homologue jointly modulate photoperiodic flowering in chrysanthemum[J]. Plant, Cell & Environment, 45(5): 1442-1456. [16] Jensen E C.2013. Quantitative analysis of histological staining and fluorescence using Image J[J]. Anatomical Record (Hoboken), 296(3): 378-381. [17] Kumagai M H, Donson J, Della-Cioppa G, et al.1995. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA[J]. Proceedings of the National Academy of Sciences, 92(5): 1679-1683. [18] Lu J, Bai M, Ren H, et al.2017. An efficient transient expression system for gene function analysis in rose[J]. Plant Methods, 13(1): 116. [19] Mortensen S, Bernal-Franco D, Cole L F, et al.2019. EASI transformation: An efficient transient expression method for analyzing gene function in Catharanthus roseus seedlings[J]. Frontiers in Plant Science, 10: 755. [20] Murai H, Mochizuki T.2022. Virus-induced gene silencing in chrysanthemum seticuspe using the tomato aspermy virus vector[J]. Plants (Basel), 11(3): 430. [21] Park M, Baek E, Yoon J, et al.2017. The use of a tobacco mosaic virus-based expression vector system in chrysanthemum[J]. The Plant Pathology Journal, 33(4): 429-433. [22] Pogue G P, Lindbo J A, Garger S J, et al.2002. Making an ally from an enemy: Plant virology and the new agriculture[J]. Annual Review of Phytopathology, 40: 45-74. [23] Sheludko Y V, Sindarovska Y R, Gerasymenko I M, et al.2007. Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated transient expression[J]. Biotechnology and Bioengineering, 96(3): 608-614. [24] Sparkes I A, Runions J, Kearns A, et al.2006. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants[J]. Nature Protocols, 1(4): 2019-2025. [25] Tang Y, Wang F, Zhao J, et al.2010. Virus-based MicroRNA expression for gene functional analysis in plants[J]. Plant Physiology, 153(2): 632-641. [26] Tian J, Pei H, Zhang S, et al.2014. TRV-GFP: A modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function[J]. Journal of Experimental Botany, 65(1): 311-322. [27] Tsuda K, Qi Y, Nguyen L V, et al.2012. An efficient Agrobacterium-mediated transient transformation of Arabidopsis[J]. The Plant Journal, 69(4): 713-719. [28] Wang L J, Cheng H, Wang Q, et al.2021. CmRCD1 represses flowering by directly interacting with CmBBX8 in summer chrysanthemum[J]. Horticulture Research, 8(1): 79. [29] Wu H Y, Liu K H, Wang Y C, et al.2014. AGROBEST: An efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings[J]. Plant Methods, 10: 19. [30] Zhang X, Ding X, Li Z, et al.2020. Development of Tomato bushy stunt virus-based vectors for fusion and non-fusion expression of heterologous proteins in an alternative host Nicotiana excelsiana[J]. Applied Microbiology and Biotechnology, 104(19): 8413-8425.
