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| Establishment and Validation of a Non-tissue Cultured Transformation System of Hairy Roots in Aeonium arboreum and Its Cultivars |
| ZHAO Rong*, HAN Hao-Zhang, HUANG Si-Yuan, HAO Xue-Ting, WANG Yu-Hua, JIN Zhao-Hui, ZHANG Qing-Hong, LI Su-Hua, ZHANG Li-Hua |
| College of Materials and Biology, Suqian University, Suqian 223800, China |
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Abstract Aeonium arboreum and its cultivars, A. arboreum and its range of succulent cultivars in the genus Aeonium of the Crassulaceae family, are important indoor ornamental plants. The absence of a transformation system in this kind of plant restricts its molecular-level studies. This study utilized the A. arboreum cultivars A. arboreum 'Halloween', A. arboreum 'Velour European' and A. arboreum as materials. Different whorl leaves and different strains of Agrobacterium rhizogenes were used to produce hairy roots under non-tissue cultured conditions, aiming to establish a non-tissue cultured hairy root transformation system for A. arboreum and its cultivars. The results indicated that using intermediate whorl leaves and the Agrobacterium rhizogenes strain ArQual could achieve the highest hairy root induction rates, with hairy root induction rates of 93.33%, 96.67% and 83.33% for A. arboreum 'Halloween', A. arboreum 'Velour European' and A. arboreum, respectively. After infection with ArQual carrying the pBI121 vector, the hairy roots produced by the intermediate whorl leaves of the A. arboreum and its cultivars turned blue upon GUS staining. The transformation efficiency of hairy root from A. arboreum 'Halloween', A. arboreum 'Velour European' and A. arboreum were 89.93%, 82.22% and 69.80%, respectively. The anthocyanin-regulating transcription factor AaMYB113 from A. arboreum 'Halloween' was used to validate the hairy roots transformation system of the A. arboreum and its cultivars, and the results showed that compared with the hairy roots transformed with an empty vector, the hairy roots transformed with AaMYB113 exhibited significantly redder coloration. Both anthocyanin content and the expression levels of anthocyanin biosynthetic structural genes in 'Halloween' hairy roots were significantly increased (P<0.05), demonstrating the feasibility of this transformation system. This study established a non-tissue cultured transformation system of hairy roots in A. arboreum and its cultivars, providing technical support for subsequent functional gene research and molecular breeding in these plants.
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Received: 06 May 2025
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Corresponding Authors:
*19127@squ.edu.cn
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[1] 艾文莉. 2015. 超积累型东南景天的遗传转化及其对水中复合重金属的净化作用[D]. 硕士学位论文, 华中师范大学, 导师:高翔, pp. 6-18. (Ai W L.2015. The establishment of genetic transformation system in Sedum alfredii Hance and its use in clean-up of the multiple metals-polluted water[D]. Thesis for M.S., Central China Normal University, Supervisor:Gao X, pp. 6-18.) [2] 陈佳欣, 梅浩, 黄彩翔, 等. 2024. 利用转基因毛状根高效培育大豆嵌合植株的方法[J]. 植物学报, 59(1):89-98. (Chen J X, Mei H, Huang C X, et al.2024. A highly efficient method to generate chimeric soybean plant with transgenic hairy roots[J]. Bulletin of Botany, 59(1):89-98.) [3] 陈新玲, 王媛, 孙吉康, 等. 2023. 蚬壳花椒毛状根的诱导及其培养技术体系构建[J]. 植物生理学报, 59(11):2117-2125. (Chen X L, Wang Y, Sun J K, et al.2023. Induction and cultivation of hairy roots of Zanthoxylum dissitum[J]. Plant Physiology Communications, 59(11):2117-2125.) [4] 侯嘉铭, 尹彦超, 田少凯, 等. 2021. 过表达CHI基因提高甘草毛状根中黄酮类化合物含量的研究[J]. 药学学报, 56(1):319-327. (Hou J M, Yin Y C, Tian S K, et al.2021. Overexpressing of chalcone isomerase (CHI) gene enhances flavonoid accumulation in Glycyrrhiza uralensis hairy roots[J]. Acta Pharmaceutica Sinica, 56(1):319-327.) [5] 侯丽丽, 施和平, 余武, 等. 2014. 烟草毛状根多倍体诱导及其植株再生[J]. 生物工程学报, 30(4):581-594. (Hou L L, Shi H P, Yu W, et al.2014. Induction of polyploid in hairy roots of Nicotiana tabacum and its plant regeneration[J]. Chinese Journal of Biotechnology, 30(4):581-594.) [6] 胡菊, 毛美琴, 杨君, 等. 2016. 4种发根农杆菌对朱砂根组培无菌叶片毛状根诱导的影响[J]. 西北植物学报, 36(2):411-418. (Hu J, Mao M Q, Yang J, et al.2016. Four kinds of Agrobacterium rhizogenes on sterile leaves induction of Ardisia crenata sims[J]. Acta Botanica Boreali-Occidentalia Sinica, 36(2):411-418.) [7] 林凯, 张育华, 柴霞, 等. 2023. 发根农杆菌介导的星油藤毛状根遗传转化体系建立[J]. 植物生理学报, 59(2):373-382. (Lin K, Zhang Y H, Cai X, et al.2023. Establishment of an Agrobacterium rhizogenes-mediated genetic transformation system of hairy roots in sacha inchi (Plukenetia volubilis L.)[J]. Plant Physiology Communications, 59(2):373-382.) [8] 刘雪羽, 杜笑雪, 陈思源, 等. 2021. 发根农杆菌介导的光皮桦毛状根高频诱导体系及遗传转化[J]. 农业生物技术学报, 29(3):495-505. (Liu X Y, Du X X, Chen S Y, et al.2021. Agrobacterium rhizogenes mediated high frequency hairy root induction system and genetic transformation in Betula luminifera[J]. Journal of Agricultural Biotechnology, 29(3):495-505.) [9] 罗萍, 张昊楠, 徐建民, 等. 2022. 发根农杆菌介导的尾巨桉遗传转化体系的建立[J]. 植物研究, 42(3):512-520. (Luo P, Zhang H N, Xu J M, et al.2022. Establishment of Agrobacterium rhizogenes-mediated genetic transformation system of Eucalyptus urophylla × E. grandis[J]. Bulletin of Botanical Research, 42(3):512-520.) [10] 梅错, 刘志鹏. 2020. 发根农杆菌介导的箭筈豌豆毛状根遗传转化体系的建立[J]. 中国草地学报, 42(5):1-7. (Mei C, Liu Z P.2020. Agrobacterium rhizogenes-mediated transformation system of common vetch (Vicia sativa subsp. nigra)[J]. Chinese Journal of Grassland, 42(5):1-7.) [11] 任艳, 李双铃, 尹亮, 等. 2018. 发根农杆菌菌株和花生品种对发根诱导率的影响[J]. 山东农业科学, 50(3):103-106. (Ren Y, Li S L, Yin L, et al.2018. Effects of Agrobacterium rhizogenes strains and peanut varieties on induction rate of hairy roots[J]. Shandong Agricultural Sciences, 50(3):103-106.) [12] 施和平, 朱远锋, 王蓓, 等. 2014. 香石竹毛状根诱导、离体培养及其植株再生[J]. 生物工程学报, 30(11):1742-1750. (Shi H P, Zhu Y F, Wang B, et al.2014. Induction and in vitro culture of hairy roots of Dianthus caryophyllus and its plant regeneration[J]. Chinese Journal of Biotechnology, 30(11):1742-1750.) [13] 陶娜, 李茂兴, 郭华春. 2023. 发根农杆菌介导的甘薯遗传转化体系优化[J]. 生物技术通报, 39(10):175-183. (Tao N, Li M X, Guo H C.2023. Optimization of sweet ptato genetic transformation system mediated by Agrobacterium rhizogenes[J]. Biotechnology Bulletin, 39(10):175-183.) [14] 王升升, 段珍, 张吉宇. 2021. 发根农杆菌介导的白花草木樨毛状根转化体系的建立[J]. 草地学报, 29(11):2591-2599. (Wang S S, Duan Z, Zhang J Y.2021. Establishment of hairy root transformation system of Melilotus albus induced by Agrobacterium rhizogenes[J]. Acta Agrestia Sinica, 29(11):2591-2599.) [15] 王艳. 2009. 发根农杆菌介导八宝景天遗传转化体系的建立[D]. 硕士学位论文, 河北农业大学, 导师:刘桂林, 梁海永, pp. 17-22. (Wang Y.2009. Establishment of genetic transformation system of Sedum spectabile boreau by Agrobacterium rhizogenes[D]. Thesis for M.S., Hebei Agricultural University, Supervisor:Liu G L, Liang H Y, pp. 17-22.) [16] 吴梦洁, 洪家都, 李芳燕, 等. 2023. 发根农杆菌介导的闽楠遗传转化体系构建与优化[J]. 核农学报, 37(8):1516-1522. (Wu M J, Hong J D, Li F Y, et al.2023. Construction and optimization of genetic transformation system mediated by Agrobacterium rhizogenes in Phoebe bournei[J]. Journal of Nuclear Agricultural Sciences, 37(8):1516-1522.) [17] 徐洪伟. 2004. 高山红景天毛状根培养系统的建立[D]. 硕士学位论文, 东北师范大学, 导师:陆静梅, pp. 24-32. (Xu H W.2004. Establishment of hairy roots culture system of Rhodiola sachalinensis A.Bor[D]. Thesis for M.S., Northeast Normal University, Supervisor:Lu J M, pp. 24-32.) [18] 徐悦, 曹英萍, 王玉, 等. 2019. 发根农杆菌介导的菠菜毛状根遗传转化体系的建立[J]. 植物学报, 54(4):515-521. (Xu Y, Cao Y P, Wang Y, et al.2019. Agrobacterium rhizogenes-mediated transformation system of Spinacia oleracea[J]. Bulletin of Botany, 54(4):515-521.) [19] 姚庆收, 单长民, 武玉永, 等. 2014. 发根农杆菌介导的垂序商陆遗传转化体系的建立及毛状根株系生物量分析[J]. 时珍国医国药, 25(8):1991-1994. (Yao Q S, Shan C M, Wu Y Y, et al.2014. Establishment of Phytolacca americana L. genetic transformation system by Agrobacterium rhizogenes and analysis of hairy root strains biomass[J]. Lishizhen Medicine and Materia Medica Research, 25(8):1991-1994.) [20] 于一凡, 郭娟, 苏平, 等. 2018. 灯盏细辛转基因毛状根体系建立[J]. 中国中药杂志, 43(10):2048-2052. (Yu Y F, Guo J, Su P, et al.2018. Establishment of a genetic transformation system for hairy root culture of Erigeron breviscapus[J]. China Journal of Chinese Materia Medica, 43(10):2048-2052.) [21] 詹玉洁, 刘博文, 张仟, 等. 2021. 发根农杆菌介导白羽扇豆转基因过表达体系的建立[J]. 植物生理学报, 57(3):655-660. (Zhan Y J, Liu B W, Zhang Q, et al.2021. Establishment of Agrobacterium rhizogenes-mediated gene overexpression system in white lupin[J]. Plant Physiology Communications, 57(3):655-660.) [22] 张弘弛, 刘瑞, 高志慧, 等. 2022. 恒山黄芪毛状根遗传转化体系的建立及活性成分含量测定[J]. 北方园艺, 9:93-99. (Zhang H C, Liu R, Gao Z H, et al.2022. Agrobacterium rhizogenes mediated transformation system of Hengshan Astragalus membranaceus and determination of active components[J]. Northern Horticulture, 9:93-99.) [23] 赵荣, 李素华, 韩浩章, 等. 2024. 多肉植物法师类品种qRT-PCR内参基因筛选[J]. 植物生理学报, 60(04):714-724. (Zhao R, Li S H, Han H Z, et al.2024. Selection of qRT-PCR internal reference gene for succulent plants of Aeonium series[J]. Plant Physiology Communications, 60(04):714-724.) [24] 赵兴坤, 孙昊, 杨丽云, 等. 2023. 发根农杆菌介导的细茎柱花草毛状根转化体系的建立[J]. 草地学报, 31(2):581-586. (Zhao X K, Sun H, Yang L Y, et al.2023. Establishment of the genetic transformation system of Stylosanthes gracilis mediated with Agrobacterium rhizogenes[J]. Acta Agrestia Sinica, 31(2):581-586.) [25] Guimaraes L A, Pereira B M, Araujo A C G, et al.2017. Ex vitro hairy root induction in detached peanut leaves for plant-nematode interaction studies[J]. Plant Methods, 13:25. [26] Hopkins D L, Durbin R D.1971. Induction of adventitious roots by culture filtrates of the hairy root bacterium, Agrobacterium rhizogenes[J]. Canadian Journal of Microbiology, 17(11):1409-1412. [27] Jedličková V, Štefková M, Mandáková T, et al.2024. Injection-based hairy root induction and plant regeneration techniques in Brassicaceae[J]. Plant Methods, 20(1):29. [28] Li C, Wang M.2021. Application of hairy root culture for bioactive compounds production in medicinal plants[J]. Current Pharmaceutical Biotechnology, 22(5):592-608. [29] Lin J, Wi D, Ly M, et al.2023. Soybean hairy root transformation for the analysis of gene function[J]. Jove-Journal of Visualized Experiments, 195:65485. [30] Lu J, Li S, Deng S, et al.2024. A method of genetic transformation and gene editing of succulents without tissue culture[J]. Plant Biotechnology Journal, 22(7):1981-1988. [31] Malarz J, Michalska K, Yudina Y V, et al.2022. Hairy root cultures as a source of polyphenolic antioxidants:Flavonoids, stilbenoids and hydrolyzable tannins[J]. Plants (Basel), 11(15):1950. [32] Ramasamy M, Dominguez M M, Irigoyen S, et al.2023. Rhizobium rhizogenes-mediated hairy root induction and plant regeneration for bioengineering citrus[J]. Plant Biotechnology Journal, 21(9):1728-1730. [33] Ron M, Kajala K, Pauluzzi G, et al.2014. Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model[J]. Plant Physiology, 166(2):455-469. [34] Tepfer M, Casse-Delbart F.1987. Agrobacterium rhizogenes as a vector for transforming higher plants[J]. Microbiological Sciences, 4(1):24-28. [35] Wang M, Qin Y Y, Wei N N, et al.2023. Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation in citrus seeds and its application in gene functional analysis[J]. Frontiers in Plant Science, 14:1293374. [36] Zhang W, Ning G, Lv H, et al.2009. Single MYB-type transcription factor AtCAPRICE:A new efficient tool to engineer the production of anthocyanin in tobacco[J]. Biochemical and Biophysical Research Communications, 388(4):742-747. [37] Zhao R, Li S H, Zhang L H, et al.2023. Transcriptome analysis reveals the effect of light on anthocyanin synthesis in leaves of Aeonium arboreum 'Halloween'[J]. Russian Journal of Plant Physiology, 70:64. [38] Zhao R, Han H Z, Li S H, et al.2024. Functional identification of AaMYB113 and AaMYB114 from Aeonium arboreum 'Halloween' in model plants[J]. Gene, 927:148699. |
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